Pharmaceutical preparations of glutathione and methods of administration thereof

ABSTRACT

A method for the administration of glutathione orally comprising the administration of a bolus of glutathione which is pharmaceutically stabilized and encapsulated. The glutathione is administered on an empty stomach. The preferred stabilizer is ascorbic acid.

FIELD OF THE INVENTION

The present invention relates to the field of antioxidant administrationto mammals, and more particularly to the field of glutathione therapiesas sole and combination therapies for mammals in need of such treatment.

BACKGROUND OF THE INVENTION

The ubiquitous tripeptide L-glutathione (GSH)(gamma-glutamyl-cysteinyl-glycine), is a well known biologicalantioxidant, and in fact is believed to be the primary intracellularantioxidant for higher organisms. When oxidized, it forms a dimer(GSSG), which may be recycled in organs having glutathione reductase.Glutathione may be transported through membranes by the sodium-dependentglutamate pump. Tanuguchi, N., et al. Eds., Glutathione Centennial,Academic Press, New York (1989), expressly incorporated herein byreference.

GSH is known to function directly or indirectly in many importantbiological phenomena, including the synthesis of proteins and DNA,transport, enzyme activity, metabolism, and protection of cells fromfree-radical mediated damage. GSH is one of the primary cellularantioxidants responsible for maintaining the proper oxidation statewithin the body. GSH is synthesized by most cells, and is also suppliedin the diet. GSH has been shown to recycle oxidized biomolecules back totheir active, reduced forms.

Reduced glutathione (GSH) is, in the human adult, produced from oxidizedglutathione (GSSG) primarily by the liver, and to a smaller extent, bythe skeletal muscle, red blood cells, and white cells. About 80% of the8-10 grams glutathione produced daily is produced by the liver anddistributed through the blood stream to the other tissues.

A deficiency of glutathione in cells may lead to excess free radicals,which cause macromolecular breakdown, lipid peroxidation, buildup oftoxins, and ultimately cell death. Because of the importance ofglutathione in preventing this cellular oxidation, glutathione iscontinuously supplied to the tissues. However, under certain conditions,the normal, physiologic supplies of glutathione are insufficient,distribution inadequate or local oxidative demands too high to preventcellular oxidation. Under certain conditions, the production of anddemand for glutathione are mismatched, leading to insufficient levels onan organismal level. In other cases, certain tissues or biologicalprocesses consume glutathione so that the intracellular levels aresuppressed. In either case, by increasing the serum levels ofglutathione, increased amounts may be directed into the cells. Infacilitated transport systems for cellular uptake, the concentrationgradient which drives uptake is increased.

As with all nutrients, it would normally be considered to eat or orallyingest the nutrient to increase body levels. Thus, attempts at oralglutathione treatments were known, and indeed the present inventorshereof previously suggested oral glutathione administration for variousindications. The protocols for administration of glutathione, however,were not optimized and therefore the bioavailability of the glutathionewas unassured and variable. All prior pharmaceutical attempts by othersto safely, effectively and predictably raise intracellular GSH throughoral therapy with GSH have not met with demonstrated success. Expertsgenerally believe that beneficial physiological effects of orallyadministered glutathione are difficult or impossible to achieve, or theefficiency is so low as to make supplementation by this routeunproductive.

Because of the poor or variable results obtained, the art generallyteaches that oral administration of glutathione is ineffective, forcingadministration or supplementation by other routes, principallyintravenously, but also by alveolar inhalation. Orally absorbed prodrugsand precursors have also been proposed or used. A known pharmacologicalregimen provides intravenous glutathione in combination with anotheragent, such as cis-platinum (a free radical associated metal drug),doxorubicin, or daunorubicin (free radical associated drugs whichinteract with nucleic acid metabolism), which produced toxic sideeffects related to free radical reactions.

The ability to harness GSH, which is a powerful, but safe substance,into an effective oral pharmaceutical had not been accomplished in thepast, because of molecular instability, poor gastrointestinal absorptionthrough existing protocols and resulting inability to reliably effectincreases in intracellular GSH levels. Administering sufficient amountsto achieve physiological benefit using known oral administrationprotocols might lead to cysteine related kidney stones, gastric distressor flatulence.

Glutathione is relatively unstable in alkaline or oxidativeenvironments, and is not absorbed by the stomach. It is believed thatglutathione is absorbed, after oral administration, if at all, in thelatter half of the duodenum and the beginning of the jejunum. It wasalso believed that orally administered glutathione would tend to bedegraded in the stomach, and that it is particularly degraded underalkaline conditions by desulfurases and peptidases present in theduodenum. Thus, known protocols for oral administration of glutathioneinvolved administered with meals or after eating to buffer pH extremesand dilute degradative enzymes. This protocol, however, has the effectof diluting the glutathione and delaying absorption. Studies directed atdetermining the oral bioavailability of glutathione under suchcircumstances showed poor absorption, and therefore such administrationwas seen as of little benefit.

Therefore, while oral dosage forms of glutathione were known, theclinical benefits of these formulations were unproved and, given thelack of predictability of their effect, these formulations were not usedfor the treatment of specific conditions, nor proven to have effect.Further, the known protocols for administration of glutathione did notprovide convenience and high bioavailability.

The prior art thus suggests that glutathione esters might be suitable asorally bioavailable sources of glutathione, which are stable and may berapidly absorbed. However, these are both more expensive thanglutathione itself and have proven toxic.

Pure glutathione forms a flaky powder which retains a static electricalcharge, due to triboelectric effects, that makes processing difficult.The powder may also have an electrostatic polarization, which is akin toan electret. Glutathione is a strong reducing agent, so thatautooxidation occurs in the presence of oxygen or other oxidizingagents. U.S. Pat. No. 5,204,114, Demopoulos et al., expresslyincorporated herein by reference in its entirety, provides a method ofmanufacturing glutathione tablets and capsules by the use of crystallineascorbic acid as an additive to reduce triboelectric effects whichinterfere with high speed equipment and maintaining glutathione in areduced state. A certain crystalline ascorbic acid is, in turn,disclosed in U.S. Pat. No. 4,454,125, Demopoulos, expressly incorporatedby reference herein in its entirety. This crystalline form is useful asa lubricating agent for machinery. Ascorbic acid has the advantage thatit is well tolerated, antioxidant, and reduces the net static charge onthe glutathione.

In synthesizing glutathione in the body, cysteine, a thiol amino acid isrequired. Since the prior art suggests that oral administration ofglutathione itself would be ineffective, prodrugs or precursor therapywas advocated. Therefore, the prior art suggests administration ofcysteine, or a more bioavailable precursor of cysteine, N-acetylcysteine (NAC). While cysteine and NAC are both, themselves,antioxidants, their presence competes with glutathione for resources incertain reducing (GSH recycling) pathways. Since glutathione is aspecific substrate for many reducing pathways, the loading of a hostwith cysteine or NAC may result in less efficient utilization orrecycling of glutathione. Thus, cysteine and NAC are not ideal GSHprodrugs. Thus, while GSH may be degraded, transported as amino acids,and resynthesized in the cell, there may also be circumstances where GSHis transported into cells without degradation; and in fact theadministration of cysteine or cysteine precursors may interfere withthis process.

A number of disease states have been specifically associated withreductions in glutathione levels. Depressed glutathione levels, eitherlocally in particular organs, or systemically, have been associated witha number of clinically defined diseases and disease states. Theseinclude HIV/AIDS, diabetes and macular degeneration, all of whichprogress because of excessive free radical reactions and insufficientGSH. Other chronic conditions may also be associated with GSHdeficiency, including heart failure and coronary artery restenosis postangioplasty.

For example, diabetes afflicts 8% of the United States population andconsumes nearly 15% of all United States healthcare costs. HIV/AIDS hasinfected nearly 1 million Americans. Current therapies cost in excess of$20,000 per year per patient, and are rejected by, or fail in 25% to 40%of all patients. Macular degeneration presently is considered incurable,and will afflict 15 million Americans by 2002.

Clinical and pre-clinical studies have demonstrated the linkage betweena range of free radical disorders and insufficient GSH levels. Newlypublished data implies that diabetic complications are the result ofhyperglycemic episodes that promote glycation of cellular enzymes andthereby inactivate GSH synthetic pathways. The result is GSH deficiencyin diabetics, which may explain the prevalence of cataracts,hypertension, occlusive atherosclerosis, and susceptibility toinfections in these patients.

GSH functions as a detoxicant by forming GSH S-conjugates withcarcinogenic electrophiles, preventing reaction with DNA, and chelationcomplexes with heavy metals such as nickel, lead, cadmium, mercury,vanadium, and manganese. GSH also plays a role in metabolism of variousdrugs, such as opiates. It has been used as an adjunct therapy totreatment with nephrotoxic chemotherapeutic agents such as cisplatin,and has been reported to prevent doxorubicin-induced cardiomyopathy. GSHis also an important factor in the detoxification of acetaminophen andethanol, two powerful hepatotoxins.

(1) HIV

High GSH levels have been demonstrated to be necessary for properfunctioning of platelets, vascular endothelial cells, macrophages,cytotoxic T-lymphocytes, and other immune system components. Recently ithas been discovered that HIV-infected patients exhibit low GSH levels inplasma, in other fluids, and in certain cell types like macrophages,which does not appear to be due to defects in GSH synthesis. GSH hasbeen shown to inhibit HIV replication in chronically-infected cells andin cells acutely infected in vitro. This makes GSH replacement therapyattractive, because it has the potential to interfere with theexpression of the integrated HIV genome, a site that is not attacked bythe currently employed antiretrovirals (AZT, ddI, ddC, D4T). GSH mayalso have benefits in countering the excess free radical reactions inHIV infection, which may be attributable to: 1) the hypersecretion ofTNF-α by B-lymphocytes, in HIV infection, and 2) the catalysis ofarachidonic acid metabolism by the gp 120 protein of HIV. Thephysiologic requirements for GSH by key cell types of the immune system,and the ability of macrophages to take up intercellular GSH, as well asto metabolically interact with T-lymphocytes to indirectly cause theirGSH to increase, offer additional reasons to attempt to correct the GSHdeficiency in HIV/AIDS.

In other new data dealing with HIV infections, the March 1997 issue ofthe Proceedings of the National Academy of Sciences (PNAS) established ". . . GSH deficiency as a key determinant of survival in HIV disease . .. " GSH deficiency is associated with impaired survival in HIV disease(PNAS. Vol. 94, pp. 1967-1972). The quest to raise GSH levels in cellsis widely recognized as being extremely important in HIV/AIDS and otherdisorders, because the low cellular GSH levels in these diseaseprocesses permit more and more free radical reactions to propel thedisorders.

HIV is known to start pathologic free radical reactions which lead tothe destruction of GSH, as well as exhaustion of other antioxidantsystems and destruction of cellular organelles and macromolecules. Inpre-clinical studies, GSH stops the replication of the virus at a uniquepoint, and specifically prevents the production of toxic free radicals,prostaglandins, TNF-α, interleukins, and a spectrum of oxidized lipidsand proteins that are immunosuppressive, cause muscle wasting andneurologic symptoms. Restoring GSH levels could slow or stop thediseases progression, safely and economically.

In mammalian cells, oxidative stresses, i.e., low intracellular levelsof reduced GSH, and relatively high levels of free radicals, activatecertain cytokines, including NFκB and TNF-α, which, in turn, activatecellular transcription of the DNA to mRNA, resulting in translation ofthe mRNA to a polypeptide sequence. In a virus infected cell, the viralgenome is transcribed, resulting in viral RNA production, generallynecessary for viral replication of RNA viruses and retroviruses. Theseprocesses require a relatively oxidized state of the cell, a conditionwhich results from stress, low glutathione levels, or the production ofreduced cellular products. The mechanism which activates cellulartranscription is evolutionarily highly conserved, and therefore it isunlikely that a set of mutations would escape this process, or that anorganism in which mutated enzyme and receptor gene products in thispathway would be well adapted for survival. Thus, by maintaining arelatively reduced state of the cell (redox potential), viraltranscription, a necessary step in late stage viral replication, isimpeded.

The amplification effect of oxidative intracellular conditions on viralreplication is compounded by the actions of various viruses and viralproducts which degrade GSH. For example, GP-120, an HIV surfaceglycoprotein having a large number of disulfide bonds, and normallypresent on the surface of infected cells, oxidizes GSH, resulting inreduced intracellular GSH levels. On the other hand, GSH reducesdisulfide bonds of GP-120, reducing or eliminating its biologicalactivity, necessary for viral infectivity. GSH therefore interferes withthe production of such oxidized proteins, and degrades them once formed.In a cell which is actively replicating viral gene products, a cascadeof events may occur which allow the cell to pass from a relativelyquiescent stage with low viral activity to an active stage with massiveviral replication and cell death, accompanied by a change in redoxpotential; by maintaining adequate GSH levels, this cascade may beimpeded.

Thus, certain viral infections, such as HIV, are associated with reducedGSH levels, and it is believed that by increasing intracellular GSHlevels in infected cells, as well as increasing extracellular GSH, thereplication of HIV may be interfered with, and the cascade of eventsdelayed or halted. It is noted that AIDS may also be associated withreduced GSSG levels, implying an interference with de novo synthesis ofGSH as well as the oxidation of existing GSH discussed above.

The Human Immunodeficiency Virus (HIV) is transmitted through twopredominant routes, contaminated blood and/or sexual intercourse. Inpediatric cases, approximately one half are infected in utero, and onehalf at delivery. This circumstance permits a study of prevention oftransmission since the time of spread is known. Initially, there is anintense viral infection simulating a severe case of the flu, withmassive replication of the virus. This acute phase passes within weeks,spontaneously, as the body mounts a largely successful immune defense.Thereafter, the individual has no outward manifestations of theinfection. However, the virus continues to replicate, insidiously,within immune system tissues and cells, like lymph nodes, lymphoidnodules and special multidendritic cells that are found in various bodycavities.

This infection is not just a viral problem. The virus, in addition toreplicating, causes excessive production of various free radicals andvarious cytokines in toxic or elevated levels. The latter are normallyoccurring biochemical substances that signal numerous reactions, usuallyexist in minuscule concentrations. Eventually, after an average of 7-10years, of seemingly quiescent HIV infection, the corrosive free radicalsand the toxic levels of cytokines begin to cause symptoms, and failuresin the immune system begin. Substances like 15-HPETE areimmunosuppressive and TNF-α causes muscle wasting, among other toxicfactors. The numbers of viral particles increase and the patientdevelops the Acquired Immune Deficiency Syndrome, AIDS, which may last 2to 4 years before the individual's demise. AIDS, therefore, is notsimply a virus infection, although the viral infection is believed to bean integral part of the etiology of the disease.

HIV has a powerful ability to mutate. It is this capability that makesit difficult to create a vaccine or to develop long-term anti-viralpharmaceutical treatments. As more people continue to fail the presentcomplex regimens, the number of resistant viral strains is increasing.This is a particularly dangerous pool of HIV and poses a considerablethreat. These resistant mutants also add to the difficulties indeveloping vaccines. This epidemic infection is out of control, and thewidely popularized polypharmaceutical regimens that are aimed only atlowering the number of viruses are proving to be too complex, too toxic,too costly, and too narrow. As a result, in the past 1.5 years since theintroduction of protease inhibitors, in combination with AZT-type drugs,increasing numbers of people are failing therapy, approximately 25% andgrowing. Further, the continuing production of free radicals andcytokines that may become largely independent of the virus, perpetuatethe dysfunctions of the immune system, the gastrointestinal tract, thenervous system, and many other organs in AIDS. The published scientificliterature indicates that many of these diverse organ systemdysfunctions are due to systemic GSH deficiencies that are engendered bythe virus and its free radicals. GSH is consumed in HIV infectionsbecause it is the principal, bulwark antioxidant versus free radicals.An additional cause of erosion of GSH levels is the presence of numerousdisulfide bonds (--S--S--) in HIV proteins, such as the GP-120 discussedabove. Disulfide bonds react with GSH and oxidize it.

This disease obviously is not controllable with the present approachesand basically can not be curtailed in its spread merely by superficialpublic health messages regarding safe sex and clean needles, or by usingoverly complex, toxic, costly medications that are narrowly aimed atjust viral replication.

The current HIV/ADS pharmaceuticals take good advantage of the conceptof pharmaceutical synergism, wherein two different targets in oneprocess are hit simultaneously. The effect is more than additive. Thedrugs now in use were selected to inhibit two very different points inthe long path of viral replication. The pathway of viral replication canbe depicted simply:

    __________________________________________________________________________    HIV Replication Pathway                                                       →                                                                              →                                                                              →                                                                              →                                                                             →                                       point #1                                                                              point #2                                                                              point #3                                                                              point #4                                                                             point #5                                       __________________________________________________________________________    Virus attacks                                                                         Virus makes                                                                           Viral DNA is                                                                          Proviral DNA                                                                         Viral RNA is                                   and enters the                                                                        DNA from its                                                                          integrated into                                                                       is inactive for a                                                                    produced, along                                cell    RNA     cells' DNA                                                                            long time, but                                                                       with viral                                                             activators will                                                                      membranes and                                                          start HIV                                                                            proteins, which                                                        replicating                                                                          are assembled                                                          rapidly                                               Viral gp120                                                                           Reverse Integrase is the                                                                      NF kappa B is                                                                        Viral protease                                 protein and                                                                           transcriptase is                                                                      enzyme  the activator of                                                                     is involved                                    CD4+ cell                                                                             the enzyme                                                                            involved                                                                              dormant HIV                                           receptors and                                                                         involved        DNA and                                               others are              glutathione                                           involved                levels must be                                                                low for                                                                       activation to                                                                 occur                                                         AZT, ddI, ddC   Glutathione                                                                          Protease                                                                      Inhibitors                                     __________________________________________________________________________

Point #2 was the earliest point of attack, using AZT-types of drugs,including ddI, ddC and others. These are toxic and eventually virusesbecome resistant to these Reverse Transcriptase inhibitors.

Point #5 is a late replication step, and this is where proteaseinhibitors function. The drug blocks viral protease, an enzyme thatsnips long protein chains to just the right length so the viral coatfits exactly around the nucleic acid core, and that proteins havingdifferent biological activities are separated. By themselves, proteaseinhibitors foster the rapid development of resistant, mutant strains.

By combining Reverse Transcriptase inhibitors plus protease inhibitors,synergism was obtained and the amounts of viral particles in the plasmaplummeted, while the speed of the developing mutant resistant viralstrains was slowed, compared to using only one type of inhibitor. Thiscombination has been in use for about 1.5 years, and so far, about 25%to 40% of U.S. patients have failed the treatment. This number isexpected to rise as resistant mutants develop, albeit more slowly thanthe use of the drugs separately.

In addition to the multiple drugs aimed at the virus, at points #2 and#5, AIDS patients and progressing HIV positive people who have not yetdeveloped an AIDS-related disease, also take other pharmaceuticals, themost common being one to prevent the unusual pneumonia caused byPneumocystis carinii, for example trimethoprim-sulfathiazole. As otheropportunistic infections occur with fungi, yeasts, bacteria,tuberculosis, and other viruses like cytomegalovirus infection of theretinae, the number of pharmaceuticals increases greatly. Sadly, AIDSpatients are also more likely to develop cancers, such as lymphomas,cancer of the cervix and Kaposi's sarcoma. Management of the cancersrequires the addition of still more drugs.

New therapies include additional drugs in the classes of ReverseTranscriptase inhibitors and protease inhibitors. Also, drugs are indevelopment to block point #3, wherein the enzyme, integrase, integratesthe HIV DNA into the infected cell's DNA, analogous to splicing a smalllength of wire into a longer wire. Vaccine development also continues,although prospects seem poor because HIV appears to be a moving targetand seems to change as rapidly as a chameleon. Vaccine development isalso impaired by the immune cell affinity of the virus.

Human Immunodeficiency virus-infected individuals have lowered levels ofserum acid-soluble thiols and GSH in plasma, peripheral blood monocytes,and lung epithelial lining fluid. In addition, it has been shown thatCD4+ and CD8+ T cells with high intracellular GSH levels are selectivelylost as HIV infection progresses. This deficiency may potentiate HIVreplication and accelerate disease progression, especially inindividuals with increased concentrations of inflammatory cytokinesbecause such cytokines stimulate HIV replication more efficiently inGSH-depleted cells. GSH and glutathione precursors such as N-acetylcysteine (NAC) can inhibit cytokine-stimulated HIV expression andreplication in acutely infected cells, chronically infected cells, andin normal peripheral blood mononuclear cells.

It is noted that depletion of GSH is also associated with a processesknown as apoptosis, or programmed cell death. Thus, intercellularprocesses which artificially deplete GSH may lead to cell death, even ifthe process itself is not lethal.

2) Diabetes Mellitus

Diabetes mellitus is found in two forms, childhood or autoimmune (typeI, IDDM) and late-onset or non-insulin dependent (type II, NIDDM). Theformer constitute about 30% and the remainder represent the bulk ofcases seen. Onset is generally sudden for Type I, and insidious for TypeII. Symptoms include excessive urination, hunger and thirst with a slowsteady loss of weight in the first form. Obesity is often associatedwith the second form and has been thought to be a causal factor insusceptible individuals. Blood sugar is often high and there is frequentspilling of sugar in the urine. If the condition goes untreated, thevictim may develop ketoacidosis with a foul-smelling breath similar tosomeone who has been drinking alcohol. The immediate medicalcomplications of untreated diabetes can include nervous system symptoms,and even diabetic coma.

Because of the continuous and pernicious occurrence of hyperglucosemia(very high blood sugar levels), a non-enzymatic chemical reaction occurscalled glycation. Since glycation occurs far more frequently insidecells, the inactivation of essential enzyme proteins happens almostcontinually. One of the most critical enzymes, γ-glutamyl-cysteinesynthetase, is glycated and readily inactivated. This enzyme is thecrucial step in the biosynthesis of glutathione in the liver.

The net result of this particular glycation is a deficiency in theproduction of GSH in diabetics. Normally, adults produce 8-10 gramsevery 24 hours, and it is rapidly oxidized by the cells. GSH is in highdemand throughout the body for multiple, essential functions, forexample, within all mitochondria, to produce chemical energy called ATP.Brain cells, heart cells, and others simply will not function well andcan be destroyed through apoptosis.

GSH is the major antioxidant in the human body and the only one we areable to synthesize, de novo. It is also the most common small molecularweight thiol in both plants and animals. Without GSH the immune systemcannot function, and the central and peripheral nervous systems becomeaberrant and then cease to function. Because of the dependence on GSH asthe carrier of nitric oxide, a vasodilator responsible for control ofvascular tone, the cardiovascular system does not function well andeventually fails. Since all epithelial cells seem to require GSH, theintestinal lining cells don't function properly and valuablemicronutrients are lost, nutrition is compromised, and microbes aregiven portals of entry to cause infections.

The use of GSH precursors cannot help to control the GSH deficiency dueto the destruction of the rate-limiting enzyme by glycation. As GSHdeficiency becomes more profound, the well-known sequellae of diabetesprogress in severity. The complications described below are essentiallydue to runaway free radical damage since the available GSH supplies indiabetics are insufficient.

The diabetic will become more susceptible to infections because theimmune system approaches collapse when GSH levels fall . . . analogousto HIV/AIDS. Peripheral vasculature becomes compromised and blood supplyto the extremities is severely diminished because GSH is not availablein sufficient amounts to stabilize the nitric oxide (•NO) to effectivelyexert its vascular dilation (relaxation) property. Gangrene is a commonsequel and successive amputations are often the result in later years.

Peripheral neuropathies, the loss of sensation commonly of the feet andlower extremities develop, often followed by aberrant sensations likeburning or itching which can't be controlled. Retinopathy andnephropathy are later events which are actually due to microangiopathy,excessive budding and growth of new blood vessels and capillaries, whichoften will bleed due to weakness of the new vessel walls. This bleedingcauses damage to the retina and kidneys with resulting blindness andrenal shutdown, the latter results in required dialysis. Cataracts occurwith increasing frequency as the GSH deficiency deepens.

Large and medium sized arteries become sites of accelerated, severeatherosclerosis, with myocardial infarcts at early ages, and of a moresevere degree. If diabetics go into heart failure, their mortality ratesat one year later are far greater than in non-diabetics. Further, ifcoronary angioplasty is used to treat their severe atherosclerosis,diabetics are much more likely to have renarrowing of cardiac vessels,termed restenosis.

The above complications are due, in large measure, to GSH deficiency andongoing free radical reactions. These sequellae frequently andeventually occur despite the use of insulin injections daily that lowerblood sugar levels. Good control of blood sugar levels is difficult forthe majority of diabetics.

3) Macular Degeneration

Approximately 1 million people in the United States have significantmacular degeneration. One out of every 4 persons aged 55 or above nowhas macular degeneration and 1 in 2 above the age of 80. As ourpopulation ages this principal cause of blindness in the elderly willincrease as well. By the year 2002, 15 million people in the U.S. willsuffer from macular degeneration.

Age-related macular degeneration (ARMD) is the disease characterized byeither a slow (dry form) or rapid (wet form) onset of destruction andirrevocable loss of rods and cones in the macula of the eye. The maculais the approximate center of the retina wherein the lens of the eyefocuses its most intense light. The visual cells, known as the rods andcones, are an outgrowth and active part of the central nervous system.They are responsible and essential for the fine visual discriminationrequired to see clear details such as faces and facial expression,reading, driving, operation of machinery and electrical equipment andgeneral recognition of surroundings. Ultimately, the destruction of therods and cones leads to functional, legal blindness. Since there is noovert pain associated with the condition, the first warnings of onsetare usually noticeable loss of visual acuity. This may already signallate stage events. It is now thought that one of the very first eventsin this pathologic process is the formation of a material called"drusen".

Drusen first appears as either patches or diffuse drops of yellowmaterial deposited upon the surface of the retina in the macula lutea oryellow spot. This is the area of the retina where sunlight is focused bythe lens. It is the area of the retina which contains the highestdensity of rods for acuity. Although cones, which detect color are lostas well in this disease, it is believed to be loss of rods which causesthe blindness. Drusen has been chemically analyzed and found to becomposed of a mixture of lipids much of it peroxidized by free radicalreactions. The Drusen first appears as small collections of material atthe base of Bruch's membrane. This produces "bubbles" which push thefirst layer of cells up off the membrane. Vascular budding, neovasculargrowth, first appears in these channels. This first layer of cells isunique.

They are retinal pigmented epithelial (RPE) cells and these cells aredistantly related to CNS microglia and have a phagocytic function. Theyare also the layer of cells immediately below the primary retinal cells,the rods and cones. The RPE cells are believed to serve a protectivefunction for the rods and cones since they consume the debris cast offby the rods and cones. It is not known yet whether the pigmentedmaterial serves a protective function or is related to phagocytosisonly. However, this pigment although concentrated in organelles, isbelieved to be composed of peroxidized lipids and melanin.

It is believed, because of the order of events in model systems, thatthe loss of RPE cells occurs first in ARMD (Age Related MacularDegeneration). Once an area of the retinal macula is devoid of RPEcells, loss of rods, and eventually some cones, occurs. Finally, buddingof capillaries begins and we see the typical microangiopathy associatedwith late stage ARMD. It is also known that RPE cells require largequantities of GSH for their proper functioning. When GSH levels dropseverely in these cells, in cell cultures where they can be studied,these cells begin to die. When cultures of these cells are supplementedwith GSH in the medium, they thrive. There is increasing evidence thatprogression of the disease is paced by a more profound deficiency in GSHwithin the retina and probably within these cells, as indicated by cellculture studies.

It is generally believed that "near" ultraviolet (UVB) and visual lightof high intensity primarily from sunlight is a strong contributingfactor of ARMD. People with light-colored irises constitute a populationat high risk, as do those with jobs which leave them outdoors and inequatorial areas where sunlight is most intense. Additional free radicalinsults, like smoking, adds to the risk of developing ARMD.

Several approaches have been recently tested, including chemotherapy,without success. Currently, there is no effective therapy to treat ARMD.Laser therapy has been developed which has been used widely to slow thedamage produced in the slow onset form of the disease by cauterizingneovascular growth. However the eventual outcome of the disease, once ithas started to progress, is certain.

Metabolism of Glutathione.

The synthesis of GSH is dependent upon the availability of cysteineeither supplied directly from the diet or cysteine or indirectly frommethionine via the transsulfuration pathway. GSH synthesis andmetabolism is governed by the enzymes of the γ-glutamyl cycle as shownin FIG. 1. GSH is synthesized intracellularly by the consecutive actionsof γ-glutamylcysteinyl synthetase (Reaction 1) and GSH synthetase(Reaction 2). The action of the latter enzyme is feedback inhibited byGSH. The breakdown of GSH (and also of its oxidized form, GSSG) iscatalyzed by γ-glutamyl transpeptidase, which catalyzes the transfer ofthe gamma-glutamyl moiety to acceptors such as sulfhydryl-containingamino acids, certain dipeptides, and GSH itself (Reaction 3). Thecellular turnover of GSH is associated with its transport, in the formof GSH, across cell membranes, where the majority of the transpeptidaseis found. During this transport, GSH interacts with γ-glutamyltransferase (also known as transpeptidase) to form γ-glutamyl aminoacids which are transported into cells. Intracellular γ-glutamyl aminoacids are substrates of γ-glutamyl cyclotransferase (Reaction 4) whichconverts these compounds into the corresponding amino acids and5-oxo-L-proline. The ATP-dependent conversion of 5-L-oxoproline toL-glutamate is catalyzed by the intracellular enzyme 5-oxo-prolinase(Reaction 5). The cysteinylglycine formed in the transpeptidase reactionis split by dipeptidase (Reaction 6). These six reactions constitute theγ-glutamyl cycle, which accounts for the synthesis and enzymaticdegradation of GSH.

Two of the enzymes of the cycle also function in the metabolism ofS-substituted GSH derivatives, which may be formed nonenzymatically byreaction of GSH with certain electrophilic compounds or by GSHS-transferases Reaction 7). The γ-glutamyl moiety of such conjugates isremoved by the action of γ-glutamyl transpeptidase (Reaction 3), areaction facilitated by γ-glutamyl amino acid formation. The resultingS-substituted cysteinylglycines are cleaved by dipeptidase (Reaction 6A)to yield the corresponding S-substituted cysteines, which may undergoN-acetylation (Reaction 8) or an additional transpeptidation reaction toform the corresponding γ-glutamyl derivative (Reaction 3A).

Intracellular GSH is converted to its oxidized, dimeric form (GSSG) byselenium-containing GSH peroxidase, which catalyzes the reduction of H₂O₂ and other peroxides (Reaction 9). GSH is also converted to GSSG bytranshydrogenation (Reaction 10). Reduction of GSSG to GSH is mediatedby the widely-distributed enzyme GSSG reductase which uses NADPH(Reaction 11). Extracellular conversion of GSH to GSSG has also beenreported; the overall reaction requires O₂ and leads to the formation ofH₂ O₂ (Reaction 12). GSSG is also formed by reaction of GSH with freeradicals.

Transport of Glutathione.

The intracellular level of GSH in mammalian cells is in the range of0.5-10 millimolar, while micromolar concentrations are typically foundin blood plasma. Intracellular glutathione is normally over 99% reducedform (GSH). The normal healthy adult human liver synthesizes 8-10 gramsof GSH daily. Normally, there is an appreciable flow of GSH from liverinto plasma. The major organs involved in the inter-organ transport ofGSH are the liver and the kidney, which is the primary organ forclearance of circulating GSH. It has been estimated to account for50-67% of net plasma GSH turnover. Several investigators have found thatduring a single pass through the kidney, 80% or more of the plasma GSHis extracted, greatly exceeding the amount which could be accounted forby glomerular filtration. While the filtered GSH is degraded stepwise bythe action of the brush-border enzymes γ-glutamyltransferase andcysteinylglycine dipeptidase, the remainder of the GSH appears to betransported via an unrelated, Na+-dependent system present inbasal-lateral membranes.

GSH transported from hepatocytes interacts with the transpeptidase ofductile cells, and there appears to be a substantial reabsorption ofmetabolites by ductule endothelium. In the rat, about 12 and 4nmoles/g/min of GSH appear in the hepatic vein and bile, respectively.

Glutathione exists in plasma in four forms: reduced glutathione (GSH),oxidized glutathione (GSSG), mixed disulfide with cysteine (CySSG) andprotein bound through a sulfhydryl linkage (GSSPr). The distribution ofglutathione equivalents is significantly different than that ofcyst(e)ine, and when either GSH or cysteine is added at physiologicalconcentration, a rapid redistribution occurs. The distribution ofglutathione equivalents in rat plasma is 70.0% protein bound, with theremaining 30% apportioned as follows: 28.0% GSH, 9.5% GSSG, and 62.6% asthe mixed disulfide with cysteine. The distribution of cysteineequivalents was found to be 23% protein bound, with the remaining 77%distributed as follows: 5.9% cysteine, 83.1% cystine, and 10.8% as themixed disulfide with glutathione. Plasma thiols and disulfides are notin equilibrium, but appear to be in a steady state maintained in part bytransport of these compounds between tissues during the interorgan phaseof their metabolism. The large amounts of protein-bound glutathione andcysteine provide substantial buffering which must be considered in theanalysis of transient changes in glutathione and cysteine. Thisbuffering may protect against transient thiol-disulfide redox changeswhich could affect the structure and activity of plasma and plasmamembrane proteins. In erythrocytes, GSH has been implicated in reactionswhich maintain the native structure of hemoglobin and of enzymes andmembrane proteins. GSH is present in erythrocytes at levels 1000 timesgreater than in plasma. It functions as the major small moleculeantioxidant defense against toxic free radicals, an inevitableby-product of the erythrocytes' handling of O₂.

Glutathione and the Immune System.

The importance of thiols and especially of GSH to lymphocyte functionhas been known for many years. Adequate concentrations of GSH arerequired for mixed lymphocyte reactions, T-cell proliferation, T- and B-cell differentiation, cytotoxic T-cell activity, and natural killer cellactivity. Adequate GSH levels have been shown to be necessary formicrotubule polymerization in neutrophils. Intraperitoneallyadministered GSH augments the activation of cytotoxic T-lymphocytes inmice, and dietary GSH was found to improve the splenic status of GSH inaging mice, and to enhance T-cell-mediated immune responses.

The presence of macrophages can cause a substantial increase of theintracellular GSH levels of activated lymphocytes in their vicinity.Macrophages consume cystine via a strong membrane transport system, andgenerate large amounts of cysteine which they release into theextracellular space. It has been demonstrated that macrophage GSH levels(and therefore cysteine equivalents) can be augmented by exogenous GSH.T-cells cannot produce their own cysteine, and it is required by T-cellsas the rate-limiting precursor of GSH synthesis. The intracellular GSHlevel and the DNA synthesis activity in mitogenically-stimulatedlymphocytes are strongly increased by exogenous cysteine, but notcystine. In T-cells, the membrane transport activity for cystine isten-fold lower than that for cysteine. As a consequence, T-cells have alow baseline supply of cysteine, even under healthy physiologicalconditions. The cysteine supply function of the macrophages is animportant part of the mechanism which enables T-cells to shift from aGSH-poor to a GSH-rich state.

The importance of the intracellular GSH concentration for the activationof T-cells is well established. It has been reported that GSH levels inT-cells rise after treatment with GSH; it is unclear whether thisincrease is due to uptake of the intact GSH or via extracellularbreakdown, transport of breakdown products, and subsequent intracellularGSH synthesis. Decreasing GSH by 10-40% can completely inhibit T-cellactivation in vitro. Depletion of intracellular GSH has been shown toinhibit the mitogenically-induced nuclear size transformation in theearly phase of the response. Cysteine and GSH depletion also affects thefunction of activated T-cells, such as cycling T-cell clones andactivated cytotoxic T-lymphocyte precursor cells in the late phase ofthe allogenic mixed lymphocyte culture. DNA synthesis and proteinsynthesis in IL-2 dependent T-cell clones, as well as the continuedgrowth of preactivated CTL precursor cells and/or their functionaldifferentiation into cytotoxic effector cells are strongly sensitive toGSH depletion.

The activation of physiologic activity of mouse cytotoxic T-lymphocytesin vivo was found to be augmented by interperitoneal (i.p.) GSH in thelate phase but not in the early phase of the response. The injection ofGSH on the third day post immunization mediated a 5-fold augmentation ofcytotoxic activity. Dietary GSH supplementation can reverseage-associated decline of immune response in rats, as demonstrated bymaintenance of Concanavalin A stimulated proliferation of splenocytes inolder rats.

Glutathione status is a major determinant of protection againstoxidative injury. GSH acts on the one hand by reducing hydrogen peroxideand organic hydroperoxides in reactions catalyzed by glutathioneperoxidases, and on the other hand by conjugating with electrophilicxenobiotic intermediates capable of inducing oxidant stress. Theepithelial cells of the renal tubule have a high concentration of GSH,no doubt because the kidneys function in toxin and waste elimination,and the epithelium of the renal tubule is exposed to a variety of toxiccompounds. GSH, transported into cells from the extracellular medium,substantially protects isolated cells from intestine and lung areagainst t-butylhydroperoxide, menadione or paraquat-induced toxicity.Isolated kidney cells also transport GSH, which can supplementendogenous synthesis of GSH to protect against oxidant injury. HepaticGSH content has also been reported to rise, indeed to double, in thepresence of exogenous GSH. This may be due either to direct transport,as has been reported for intestinal and alveolar cells, or viaextracellular degradation, transport, and intracellular resynthesis.

The nucleophilic sulfur atom of the cysteine moiety of GSH serves as amechanism to protect cells from harmful effects induced by toxicelectrophiles. The concept that glutathione S-conjugate biosynthesis isan important mechanism of drug and chemical detoxification is wellestablished. GSH conjugation of a substrate generally requires both GSHand glutathione-S-transferase activity. The existence of multipleglutathione-S-transferases with specific, but also overlapping,substrate specificities enables the enzyme system to handle a wide rangeof compounds.

Several classes of compounds are believed to be converted by glutathioneconjugate formation to toxic metabolites. Halogenated alkenes,hydroquinones, and quinones have been shown to form toxic metabolitesvia the formation of S-conjugates with GSH. The kidney is the maintarget organ for compounds metabolized by this pathway. Selectivetoxicity to the kidney is the result of the kidney's ability toaccumulate intermediates formed by the processing of S-conjugates in theproximal tubular cells, and to bioactivate these intermediates to toxicmetabolites.

The administration of morphine and related compounds to rats and miceresults in a loss of up to approximately 50% of hepatic GSH. Morphine isknown to be biotransformed into morphinone, a highly hepatotoxiccompound, which is 9 times more toxic than morphine in mouse bysubcutaneous injection, by morphine 6-dehydrogenase activity. Morphinonepossesses an α, β-unsaturated ketone, which allows it to form aglutathione S-conjugate. The formation of this conjugate correlates withloss of cellular GSH. This pathway represents the main detoxificationprocess for morphine. Pretreatment with GSH protects againstmorphine-induced lethality in the mouse.

The deleterious effects of methylmercury on mouse neuroblastoma cellsare largely prevented by coadministration of GSH. GSH may complex withmethylmercury, prevent its transport into the cell, and increasecellular antioxidant capabilities to prevent cell damage. Methylmercuryis believed to exert its deleterious effects on cellular microtubulesvia oxidation of tubulin sulfhydryls, and by alterations due toperoxidative injury. GSH also protects against poisoning by other heavymetals such as nickel and cadmium.

Because of its known role in renal detoxification and its low toxicity,GSH has been explored as an adjunct therapy for patients undergoingcancer chemotherapy with nephrotoxic agents such as cisplatin, in orderto reduce systemic toxicity. In one study, GSH was administeredintravenously to patients with advanced neoplastic disease, in twodivided doses of 2,500 mg, shortly before and after doses ofcyclophosphamide. GSH was well-tolerated and did not produce unexpectedtoxicity. The lack of bladder damage, including microscopic hematuria,supports the protective role of this compound. Other studies have shownthat i.v. GSH coadministration with cisplatin and/or cyclophosphamidecombination therapy, reduces associated nephrotoxicity, while not undulyinterfering with the desired cytotoxic effect of these drugs.

Clinical Use of Glutathione

Ten elderly patients with normal glucose tolerance and ten elderlypatients with impaired glucose tolerance (IGT) underwent GSH infusion,10 mg/min for 120 min, for a total dose of 1,200 mg in 2 hr, under basalconditions and during 75 g oral glucose tolerance tests and intravenousglucose tolerance tests. Basal plasma total glutathione levels wereessentially the same for normal and IGT groups, and GSH infusion underbasal conditions increased GSH to similar levels. This studydemonstrated that GSH significantly potentiated glucose-induced insulinsecretion in patients with IGT. No effect was seen on insulin clearanceand action.

The antihypertensive effect of an i.v. bolus of 1,844 mg. or 3,688 mg.GSH was studied in normal and mild to moderate essential hypertensivesubjects and in both hypertensive and non-hypertensive diabetics, bothtype I and type II. The administration of 1,844 mg. GSH produced a rapidand significant decrease in both systolic and diastolic blood pressure,within ten minutes, but which returned to baseline within 30 minutes, inboth groups of hypertensive patients and in non-hypertensive diabetics,but had no effect in normal healthy subjects. At the 3,699 mg. dose, notonly did the blood pressure decrease in the hypertensive subjects, butGSH produced a significant decrease in the blood pressure values innormal subjects as well.

GSH, 1,200 mg/day intravenously administered to chronic renal failurepatients on hemodialysis was found to significantly increase studiedhematologic parameters (hematocrit, hemoglobin, blood count) as comparedto baseline, and holds promise to reverse the anemia seen in thesepatients.

Toxicological Effects of Glutathione.

The reported LD₅₀ of GSH in rats and mice via various routes ofadministration are listed in the table below. GSH has an extremely lowtoxicity, and oral LD₅₀ measurements are difficult to perform due to thesheer mass of GSH which has to be ingested by the animal in order to seeany toxic effects.

    __________________________________________________________________________        Route of                                                                  Animal                                                                            Admin.  LD.sub.50                                                                           Reference                                                   __________________________________________________________________________    Mouse                                                                             Oral    5000 mg/kg                                                                          Modern Pharmaceuticals of Japan, IV Edition,                                  Tokyo, Japan Pharmaceutical, Medical and                                      Dental Supply Exporters' Association, 1972, p                                 93.                                                         Mouse                                                                             Intraperitoneal                                                                       4020 mg/kg                                                                          Modern Pharmaceuticals of Japan, IV Edition,                                  Tokyo, Japan Pharmaceutical, Medical and                                      Dental Supply Exporters' Association, 1972, p                                 93.                                                         Mouse                                                                             Intraperitoneal                                                                       6815 mg/kg                                                                          Toxicology, vol. 62, p. 205, 1990.                          Mouse                                                                             Subcutaneous                                                                          5000 mg/kg                                                                          Modern Pharmaceuticals of Japan, IV Edition,                                  Tokyo, Japan Pharmaceutical, Medical and                                      Dental Supply Exporters' Association, 1972, p                                 93.                                                         Mouse                                                                             Intravenous                                                                           2238 mg/kg                                                                          Japanese J. of Antibiotics, vol. 38, p.137,                                   1985.                                                       Mouse                                                                             Intramuscular                                                                         4000 mg/kg                                                                          Modern Pharmaceuticals of Japan, III Edition,                                 Tokyo, Japan Pharmaceutical, Medical and                                      Dental Supply Exporters' Association, 1968, p                                 97.                                                         __________________________________________________________________________

GSH can be toxic, especially in cases of ascorbate deficiency, and theseeffects may be demonstrated in, for example, ascorbate deficient guineapigs given 3.75 mmol/kg daily (1,152 mg/kg daily) in three divideddoses, whereas in non-ascorbate deficient animals, toxicity was not seenat this dose, but were seen at double this dose.

Use of High-Dose Oral GSH in Cancer Patients.

In one published study, eight patients with hepatocellular carcinomawere treated with 5 g oral reduced glutathione per day. Two patientswithdrew shortly after receiving GSH due to intolerable side-effects(gastrointestinal irritation and sulfur odor). The remaining patients,aged 27-63, three male and three female, did not experience side-effectsfrom this high dose of GSH and continued to take 5 g oral GSH forperiods ranging from 119 days (at which time the patient died from hertumor) to >820 days (this patient was still alive at the time ofpublication and was still taking 5 g oral GSH daily; his tumor had notprogressed and his general condition was good). Two of the femalepatients survived 1 year and exhibited regression or stagnation of theirtumor growth. The remaining two patients, both male, died as expectedwithin 6 months.

Experience in HIV-Infected Patients.

A commercially available nutritional formulation containing 3 grams ofreduced glutathione was given daily to a group of 46 AIDS patients for aperiod of three months by a group of private physicians. No significantGSH-related adverse effects were reported. No evidence of toxicitiesfrom laboratory studies or from clinical examinations was reported;however, no benefit was conclusively demonstrated.

Pharmacokinetics of Glutathione

The pharmacokinetics of intravenously administered GSH were determinedin the rat and interpreted by means of an open, two-compartment model.Following a bolus injection of 50-300 mmol/kg GSH, arterial plasmaconcentrations of (i) GSH, (ii) oxidized glutathione/GSSG, (iii) totalthiols, and (iv) soluble thiols minus GSH, were elevated and thenrapidly decreased non-exponentially, as anticipated. With increasingdose, the rate constant for drug elimination and plasma clearanceincreased form 0.84 to 2.44 mL/min. and the half-life of the eliminationphase decreased from 52.4 to 11.4 minutes. Both the apparent volume ofdistribution and the degree of penetration of GSH into the tissues werediminished with increasing dose (from 3.78 to 1.33 L/Kg and from 6.0 to0.51 as k₁₂ /k₂₁, respectively). The data indicate that GSH is rapidlyeliminated. This is mainly due to rapid oxidation in plasma rather thanby increased tissue extraction or volume distribution. Thus, plasma GSHlevels appear to be quickly regulated by which the body may maintainconcentrations within narrow physiological limits.

When single doses of 600 mg GSH were administered intravenously tosheep, GSH levels in venous plasma and lung lymph rose transiently. Themean concentration was approximately 50 mM for venous plasma, peaking at30 min, and returning to baseline after 45 minutes. Lung lymph peaklevel was about 100 mM at 15 min, returning to baseline after 30minutes. Average epithelial lining fluid (ELF) levels were variable butshowed no significant increase over baseline during the three hourobservation period. Urine excretion was rapid with peak levels at 15minutes. In both plasma and lung lymph, GSH accounted for greater than95% of the total glutathione (GSH plus GSSG). In ELF 75.4% of thebaseline glutathione was in the reduced form, whereas in urine 59.6% waspresent as GSH.

Orally ingested reduced glutathione is absorbed intact from the smallintestine in a rat model, specifically in the upper jejunum. It is notedthat rat metabolism differs from man, and therefore the results of ratstudies should be verified in man before the results are extrapolated.Plasma GSH concentration in rats increased from 15 to 30 mM afteradministration of GSH either as a liquid bolus (30 mM) or mixed (2.5-50mg/g) in AIN-70 semi-synthetic diet (11). GSH concentration was maximalat 90-120 minutes after GSH administration and remained high for over 3hours. Administration of the amino acid precursors of GSH had little orno effect on plasma GSH values, indicating that GSH catabolism andre-synthesis do not account for the increased GSH concentration seen.Inhibition of GSH synthesis and degradation byL-buthionine-[S,R]-sulfoximine (BSO) and acivicin showed that theincreased plasma GSH came mostly from absorption of intact GSH insteadof from its metabolism. Plasma protein-bound GSH also increased afterGSH administration, with a time course similar to that observed for freeplasma GSH. Thus, dietary GSH can be absorbed intact and results in asubstantial increase in blood plasma GSH.

Administration of oral GSH increased hepatic glutathione levels in: (i)rats fasted 48 hours, (ii) mice treated with GSH depletors, and (iii)mice treated with paracetamol (a drug which promotes a depletion ofhepatic GSH followed by hepatic centrilobular necrosis). In theseexperiments, the animals were orally intubated with 1000 mg/kg bodyweight GSH. Mean pretreatment values in 48-hour fasted rats were 3.0-3.1mmol/g fresh hepatic tissue. Mean values after treatment were 5.8, 4.2,and 7.0 mmol/g fresh hepatic tissue for 2.5, 10, and 24 hourspost-treatment, respectively. Mice were given an oral dose of GSH (100mg/kg) and concentrations of GSH were measured at 30, 45 and 60 min inblood plasma and after 1 hr in liver, kidney, heart, lung, brain, smallintestine and skin. GSH concentrations in plasma increased from 30 mM to75 mM within 30 min of oral GSH administration, consistent with a rapidflux of GSH from the intestinal lumen to plasma. No increases overcontrol values were obtained in most tissues except lung over the sametime course. Mice pretreated with the GSH synthesis inhibitor BSO hadsubstantially decreased tissue concentrations of GSH, and oraladministration of GSH to these animals resulted instatistically-significant increases in the GSH concentrations of kidney,heart, lung, brain, small intestine and skin but not in liver.

The kinetics and the effect of glutathione on plasma and urinesulphydryls were studied in ten healthy human volunteers. Following theintravenous infusion of 2000 mg/m² of GSH the concentration of totalglutathione in plasma increased from 17.5-13.4 mmol/Liter (mean =/- SD)to 823-326 mmol/Liter. The volume of distribution of exogenousglutathione was 176-107 Ml/Kg and the elimination rate constant was0.063-0.027/minute, corresponding to a half-life of 14.1-9.2 minutes.Cysteine in plasma increased from 8.9-3.5 mmol/Liter to 114-45mmol/Liter after the infusion. In spite of the increase in cysteine, theplasma concentration of total cyst(e)ine (i.e. cysteine, cystine, andmixed disulphides) decreased, suggesting an increased uptake of cysteinefrom plasma into cells. The urinary excretion of glutathione and ofcyst(e)ine was increased 300-fold and 10-fold respectively, in the 90minutes following the infusion.

Normal healthy volunteers were given an oral dose of GSH to determinewhether dietary GSH could raise plasma GSH levels. Results showed thatan oral dose of GSH (15 mg/kg) raised plasma glutathione levels inhumans 1.5-10 fold over the basal concentration in four out of fivesubjects tested, with a mean value three times that of normal plasma GSHlevels. Plasma GSH became maximal 1 hour after oral administration,dropping to approximately 1/2 maximal values after three hours.Equivalent amounts of GSH amino acid constituents failed to increaseplasma levels of GSH. GSH bound to plasma proteins also increased withthe same time course as seen with free GSH.

SUMMARY OF THE INVENTION

The present inventors have found that oral glutathione bioavailabilityand efficiency may be increased by the administration ofpharmaceutically stabilized reduced glutathione in a bolus on an emptystomach.

The present inventors have also found that glutathione is efficientlyabsorbed from mucous membranes, especially the sublingual mucosa andlumen of the duodenum and initial part of the ileum.

As used herein, the term "pharmaceutically stabilized glutathione"refers to glutathione which is maintained in a reduced form withoutsubstantial cyclization. This stabilization may be effected by theaddition of one or more agents which, together with the glutathione,provide a pharmaceutical formulation which is capable of deliveringnative reduced glutathione.

The present invention also includes novel combinations of glutathioneand other pharmacological agents and in novel dosage forms and means foradministration.

The oral administration of pharmaceutically stabilized reducedglutathione, presented as a charge transfer complex in relatively highconcentration may produce a significant, predictable increase inintracellular glutathione levels in humans.

BRIEF DESCRIPTION OF THE DRAWINGS AND APPENDICES

The invention is shown by way of example in the drawings and appendices,in which:

FIG. 1 shows a graph of a clinical response of an HIV infected subjectto 1 gram of administered glutathione; and

FIG. 2 shows a table of clinical study results.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

It has been found that, in otherwise healthy HIV infected humans, theintracellular glutathione levels in the peripheral blood mononucleocytes(PBMs) was significantly increased after oral administration ofstabilized glutathione. This is achieved by providing a glutathioneformulation which ensures delivery of adequate dose of pharmaceuticallystabilized, reduced glutathione, with rapid dissolution before theduodenum. The formulation is administered to efficiently provide a highconcentration of glutathione in the duodenum, i.e., on an empty stomach,to enhance uptake.

A preferred formulation includes 250 mg. or more of reduced glutathionewith at least equimolar ascorbic acid, to fulfill three functions: actsas a sacrificial non-specific antioxidant during preparation, storageand after ingestion; reduces or neutralizes static electrical charge ofglutathione powder, allowing dense packing of capsules; and acts as alubricant for the encapsulation device. The ascorbic acid also maintainsan acidic and reducing environment, which pharmaceutically stabilizesthe glutathione molecule. Ascorbic acid is believed to form a chargecouple with glutathione which enhances penetration through cellmembranes, and reduces the tendency for the gamma-glutamyl and glycinylresidues to assume a cyclic conformation or to form an internal cyclicamide. The ascorbate thus complexes with the glutathione in solution tomaintain a linear conformation. This linear conformation, in turn,stericly hinders the free cysteinyl thiol group. This steric hindrancestabilizes a free radical which may be formed, and thus maintains thebiological activity of glutathione.

A cyclic form of glutathione, which may occur under certain conditions,such as neutral to basic pH, exposes the sulfhydryl moiety, making itmore reactive. Under alkaline pH, cyclic amide formation is promoted,leaving a potentially toxic compound. The cyclic glutathione compositionis a potential structural analog which may inhibit glutathionereductase, glutathione peroxidase and specific glutathione transporterproteins.

Likewise, oxidizing conditions promote disulfide formation (GSSG andPr--S--S--G), which may reduce bioavailability of glutathione andcounteract some of the potential benefits of glutathione administration.Further, oxidizing conditions also promote desulfuration, resulting inopthalmic acid formation (or other compounds), which may be toxic orinhibit efficient glutathione absorption.

A preferred oral formulation thus preferably includes an effectiveamount of glutathione mixed with a stabilizing agent, which isadministered under such conditions that the concentration of glutathioneattained in the lumen of the latter portion of the duodenum is higherthan the plasma glutathione concentration, and preferably higher thanthe intercellular concentration of the epithelial lining cells. Thus,for example, a glutathione and ascorbic acid capsule is taken on anempty stomach. The reducing agent, preferably ascorbic acid, preventsoxidation of the glutathione during packaging and storage, and furthermay stabilize the glutathione in the relatively alkaline conditions ofthe duodenum prior to absorption. Desulfuration of glutathione leads tothe formation of ophthalmic acid, the serine analog of glutathione,which inhibits glutathione uptake. This protocol is in contrast to priorart administration methods, which direct taking glutathione capsulesafter meals. By diluting glutathione with food, degradative enzymes arediluted and alkaline conditions buffered; however, according to thepresent invention, the rapidity of absorption allows highbioavailability with only a small amount of degradation.

The present invention also advantageously provides a method of use andpharmaceutical formulation of glutathione combined and anotherpharmaceutically active composition, wherein the other composition isselected from a broad group consisting of:

easily oxidized compositions,

antioxidant compositions,

compositions with oxidant effects,

compositions for the treatment of pathology associated with:

suppressed total glutathione levels,

suppressed reduced glutathione levels,

relatively oxidized conditions in the organism,

uncontrolled free radical or oxidizing reactions, or

conditions where a more reduced state is desirable.

Glutathione may be used alone or in combination with other knowncompositions for the treatment or palliation of AIDS, HIV infection orretroviral replication (e.g., HTLV I, HTLV-II, HTLV-III, etc.), herpesvirus replication (e.g., Herpes simplex type I, Herpes simplex type II,Herpes zoster (varicella), CMV, EBV, HHV-8, etc.), rabies, ebola virus,influenza virus, CHF, coronary artery disease, status post coronaryartery restenosis, Diabetes mellitus, Macular Degeneration, and/orhepatitis (toxic or infectious).

Glutatahione may also be used, alone or in combination with othertherapies for the treatment of free radical associated neurologicalconditions, for example, Alzheimer's disease, Parkinson's disease,catecholamine toxicities, other free-radical associated toxicities,stroke and transient ischemic events, spinal chord injury and othertraumatic injuries to nerve tissue, peripheral neuropathies, possiblyprion-associated illness, infectious agent pathology and inflammatorypathology, or to reduce the free-radical associated toxicity of drugsadministered to treat these conditions.

Mycoplasma infections, such as mycoplasma pneumonia, are believed tocause pathology due to free radical reactions within cells by theseintracellular parasites. Therefore, glutathione may be administeredalone or in combination with an anti-mycoplasma antibiotic for thetreatment of mycoplasma infections.

The present invention may also be used to increase or supplement theglutathione levels in normal mammals. This may be desired, for example,for prophylaxis against ischemic events, free radical damage from sun,chemicals, or other environmental exposure, and to reduce a cancer risk.

In fact, since oxidizing conditions in an organism are generallyundesirable, and where necessary the mechanisms for producing oxidizingconditions typically overpower ingested antioxidants, a large number ofmedications and drugs are appropriate for combination with glutathione.However, certain conditions may require care in the administration ofglutathione. Further, certain cancer chemotherapy regimens rely onexhaustion of cellular free radical quenching mechanisms to selectivelykill target cells. Finally, cellular apoptosis, or programmed celldeath, relies on exhaustion of reduced glutathione levels in cells(mitochondria), resulting in death. Where this mechanism is required orphysiologically correct, interruption by exogenous glutathione may beundesirable. Further, glutathione may interact with some compositions,either to non-specifically reduce or combine with the chemical moiety,or to alter a metabolism after ingestion; unless accounted for, theseeffects may be undesirable.

A known anti-HIV therapy, 3'-azidothymidine (zidovudine, AZT), acts as apotent reverse transcriptase inhibitor. This drug, however, generatesfree radicals and is toxic to mitochondria, and is associated with amyopathy. Glutathione may therefore be administered in conjunction withAZT to reduce toxicity while not interfering with the reversetranscriptase inhibitory activity, thus increasing the therapeuticindex. Likewise, glutathione may also be used to increase thetherapeutic index of other drugs which have a significant free-radicalassociated toxicity.

There are a number of conditions which are believed to be associatedwith reduced intracellular antioxidant levels, including AIDS, diabetes,macular degeneration, congestive heart failure, vascular disease andcoronary artery restenosis, Herpes virus infection, toxic and infectioushepatitis, and rabies. Certain interstitial lung disease may be due toinsufficient glutathione levels. Further, various toxins and medicationsmay also result in free radical reactions, including types of cancerchemotherapy. Therefore, the present invention holds potential to treatthese diseases and conditions by the use of a convenient, effective oralformulation of glutathione. Thus, the administration of exogenousglutathione supplements the hepatic output to help maintain reducedconditions within the organism. As noted above, the failure to quenchfree radical reactions allows an undesirable cascade resulting in damageto macromolecules, lipid peroxidation, and generation of toxiccompounds. The maintenance of adequate glutathione levels is necessaryto block these free radical reactions.

Glutathione also has the ability to form complexes with metals. Forexample, as discussed above, glutathione forms chelation complexes withnickel, lead, cadmium, mercury, vanadium and manganese. Glutathione alsoforms circulating complexes with copper in the plasma. According to thepresent invention, glutathione may be administered to treat metaltoxicity. It is believed that the glutathione-metal complexes will beexcreted, thus reducing the metal load. Thus, glutathione may beadministered for the treatment of toxicity associated with iron, copper,nickel, lead, cadmium, mercury, vanadium, manganese, cobalt, transuranicmetals, such as plutonium, uranium, polonium, and the like. As comparedto EDTA, glutathione has a reduced tendency to chelate calcium,providing a significant advantage. It is noted that the chelationproperties of glutathione are separate from the antioxidant properties;however, some metal toxicities are free radical mediated, for exampleiron, and therefore glutathione administration for these conditions isparticularly advantageous.

In order to provide high bioavailability, it has been found desirable toprovide a relatively high concentration of reduced glutathione inproximity to the mucous membrane, e.g., the duodenum for oraladministration. Thus, in contrast to prior methods, the glutathione ispreferably administered as a single bolus on an empty stomach. Thepreferred dosage is between about 100-10,000 mg. glutathione, and morepreferably between about 250-3,000 mg. glutathione. Further, theglutathione formulation is preferably stabilized with a reducing agent(antioxidant), preferably ascorbic acid, to reduce oxidation both duringstorage and in the digestive tract prior to absorption. The use ofcrystalline ascorbic acid has the added benefit of reducing the staticcharge of glutathione for improved encapsulation and serving as alubricant for the encapsulation apparatus. However, other staticdissipation methods or additives may be employed, and other antioxidantsmay be employed. The preferred dosage form is a capsule, e.g., atwo-part gelatin capsule, which protects the glutathione from air andmoisture, while dissolving quickly in the stomach.

The digestive tract is believed to have specific facilitated or activetransport carriers for glutathione which allow uptake of glutathionefrom the intestinal lumen without degradation. According to the presentinvention, the uptake through this mechanism is maximized by providing ahigh concentration gradient and avoiding the presence or production oftransport inhibitors, such as ophthalmic acid. Thus, the preferredmethod of oral administration according to the present invention employsan uptake mechanism which differs

The oral mucosa have been found to allow rapid and efficient uptake ofglutathione into the blood. In contrast to the digestive tract, thesignificance of facilitated or active transport mechanisms in the oralmucosa is believed to be low; rather, a high concentration ofglutathione in the oral mucosa is believed to permit passive transportof the glutathione through the cells or around the cells into thecapillary circulation. Therefore, compositions intended for absorptionthrough the oral mucosa, e.g., for sublingual administration, arepreferably of high purity, as contaminants may be absorbed similarly toglutathione, and as relatively small, uncharged molecules. Therefore,the composition preferably includes ascorbic acid which helps tomaintain the glutathione in a reduced state, maintains a somewhat acidicenvironment in the mouth to avoid deprotonation of the glutamic acidresidue, without causing substantially all of the amines to beprotonated.

It has been found, contrary to reports of other scientists, thatglutathione is not substantially degraded in the stomach, and therefore,the release of the glutathione need not diluted in the stomach orrelease be delayed. In fact, according to the present invention, theglutathione formulation is preferably released and dissolved in thestomach. The addition of stabilizer, i.e., ascorbic acid, furtherimproves the ability of the glutathione to reach its site of absorptionin the intestine undegraded. Once past the stomach, it is important thatthe glutathione be immediately available for absorption, as thedesulfurases and peptidases from the pancreas, as well as the increasein pH, do tend to degrade the glutathione. The desulfurase producesophthalmic acid, which interferes with glutathione absorption. Thus, byproviding a high concentration of glutathione in the duodenum, withoutsubstantial dilution, the glutathione may be absorbed at a maximum rate.While the degradation of glutathione in the latter part of the duodenumand ileum may compete with the absorption process, the present methodprovides significant bioavailability. In fact, studies have demonstratedabout 90% bioavailability of orally administered glutathione accordingto the present invention.

The capsule is preferably a standard two-part hard gelatin capsule ofdouble-O (OO) size, which may be obtained from a number of sources.After filling, the capsules are preferably stored under nitrogen, toreduce oxidation during storage. The capsules are preferably filledaccording to the method of U.S. Pat. No. 5,204,114, incorporated hereinby reference in its entirety, using crystalline ascorbic acid as both anantistatic agent and stabilizer. Further, each capsule preferablycontains 500 mg of glutathione and 250 mg of crystalline ascorbic acid.A preferred composition includes no other excipients or fillers;however, other compatible fillers or excipients may be added. Whilediffering amounts and ratios of glutathione and stabilizer may be used,these amounts are preferable because they fill a standard double-Ocapsule, and provide an effective stabilization and high dose. Further,the addition of calcium carbonate, a component of known high doseglutathione capsules, is avoided as there may be impurities in thiscomponent. Further, calcium carbonate acts as a base, neutralizingstomach acid, which accelerates degradation of glutathione in the smallintestine.

Because the glutathione and ascorbic acid are administered in relativelyhigh doses, it is preferred that these components be highly purified, toeliminate impurities, toxins or other chemicals, which may destabilizethe formulation or produce toxic effects or side effects. As statedabove, the formulation may also include other pharmaceutical agents, ofvarious classes.

Glutathione is advantageously administered over extended periods.Therefore, one set of preferred useful combinations include glutathioneand drugs intended to treat chronic conditions which are well absorbedon an empty stomach, and do not have adverse interactions or reduced orvariable combined absorption.

One particular class of drugs includes central or peripheral adrenergicor catecholenergic agonists, or reuptake blockers, which may produce anumber of toxic effects, including neurotoxicity, cardiomyopathy andother organ damage. These drugs are used, for example, as cardiac,circulatory and pulmonary medications, anesthetics andpsychotropic/antipsychotic agents. Some of these drugs also have abusepotential, as stimulants, hallucinogens, and other types ofpsychomimetics. Other free radical initiation associated drugs includethorazine, tricyclic antidipressants, quinolone antibiotics,benzodiazepines, acetaminphen and alcohol.

Therefore, it is an aspect of the present invention to provide an oralpharmaceutical formulation comprising glutathione in an amount ofbetween about 50-10,000 mg, and an effective amount of a pharmacologicalagent capable of initiating free radical reactions in a mammal. Thepharmacological agent is, for example, an adrenergic, dopaminergic,serotonergic, histaminergic, cholinergic, gabaergic, psychomimetic,quinone, quinolone, tricyclic, and/or steroid agent.

Hepatic glutathione is consumed in the metabolism, catabolism and/orexcretion of a number of agents. The depletion of hepatic glutathionemay result in hepatic damage or a toxic hepatitis. Such agents mayinclude, for example, aminoglycoside antibiotics, acetominophen,morphine and other opiates. High dose niacin, used to treathypercholesterolemia, has also been associated with a toxic hepatitis.The present invention therefore encompasses an oral pharmaceuticalformulation comprising glutathione in an amount of between about50-10,000 mg, administered in conjunction with and an effective amountof a pharmacological agent which consumes hepatic glutathione reserves.

A number of pathological conditions result in hepatic damage. Thisdamage, in turn, reduces the hepatic reserves of glutathione and theability of the liver to convert oxidized glutathione to its reducedform. Other pathological conditions are associated with impairedglutathione metabolism. These conditions include both infectious andtoxic hepatitis, cirrhosis, hepatic primary and metastatic carcinomas,traumatic and iatrogenic hepatic damage or resection. The presentinvention encompasses a pharmaceutical formulation comprisingglutathione and an antiviral or antineoplastic agent. The antiviral orantineoplastic agent is, for example, a nucleoside analog.

Glutathione is broken down, and cysteine excreted, possibly in theurine. Very high doses of glutathione may therefore result incysteinuria, which may result in cysteine stones. Other long termtoxicity or adverse actions may result. Therefore, a daily intake ofgreater than about 10 gm. for extended period should be medicallymonitored. On the other hand, individual doses below about 50 mg. areinsufficient to raise the concentration of the duodenal lumen to highlevels to produce high levels of absorption, and to provide clinicalbenefit. Therefore, the preferred formulations according to the presentinvention have glutathione content greater than 50 mg, and provided inone or more doses totaling up to about 10,000 mg per day.

In the treatment of HIV infection, it is believed that the oraladministration of a relatively high dose bolus of glutathione, i.e., 1-3grams per day, on an empty stomach, will have two beneficial effects.First, HIV infection is associated with a reduction in intracellularglutathione levels in PBMs, lung, and other tissues. It is furtherbelieved that by increasing the intracellular glutathione levels, thatthe functioning of these cells may be returned to normal. Therefore, theadministration of glutathione according to the present invention willtreat the effects of HIV infection. Therefore, the present inventionencompasses the oral administration of glutathione and ascorbic acid,optionally in combination with an antiretroviral agent. It is noted thatthe transcription mechanisms and control involved in retroviralinfection is believed to be relatively conserved between various types.Therefore, late stage retroviral suppression is expected for the varioustypes of human retroviruses and analogous animal retroviruses.

Second, it has been found in in vitro tests that by increasing theintracellular levels of glutathione in infected monocytes to the highend of the normal range, the production of HIV from these cells may besuppressed for about 35 days. This is believed to be related to theinterference in activation of cellular transcription by cytokines,including NFkB and TNFα. Therefore, the infectivity of HIV infectedpersons may be reduced, helping to prevent transmission. This reductionin viral load may also allow the continued existence of uninfected butsusceptible cells in the body.

Glutathione, administered according to the present method, is believedto be effective in the treatment of congestive heart failure (CHF). InCHF, there are believed to be two defects. First, the heart muscle isweakened, causing enlargement of the heart. Second, peripheral vasospasmis believed to be present, causing increased peripheral resistance.Glutathione is effective in enhancing the effects of nitric oxide, andtherefore is believed to be of benefit to these patients by decreasingvasoconstriction and peripheral vascular resistance, while increasingblood flow to the tissues. While nitroso-glutathione is more effectiveat preventing platelet aggregation than at vasodilation, it isnevertheless a potent vasodilator with a longer half-life than nitricoxide alone. Further, since a relative hypoxia of the tissues isassociated with free radical-mediated cellular damage, the presence ofglutathione will also help to block this damage. The present inventiontherefore encompasses the oral administration of glutathione inconjunction with a congestive heart failure medication, for example,digitalis glycosides, dopamine, methyldopa, phenoxybenzamine,dobutamine, terbutaline, amrinone, isoproterenol, beta blockers, calciumchannel blockers, such as verapamil, propranolol, nadolol, timolol,pindolol, alprenolol, oxprenolol, sotalol, metoprolol, atenolol,acebutolol, bevantolol, tolamolol, labetalol, diltiazem, dipyridamole,bretylium, phenytoin, quinidine, clonidine, procainamide, acecainide,amiodarione, disopyramide, encainide, flecanide, lorcainide, mexiletine,tocainide, captopril, minoxodil, nifedipine, albuterol, pargyline,vasodilators, including nitroprusside, nitroglycerin, phentolamine,phenoxybenzamine, hydrazaline, prazosin, trimazosin, tolazoline,trimazosin, isosorbide dinitrate, erythrityl tetranitrate, asprin,papaverine, cyclandelate, isoxsuprine, niacin, nicotinyl alcohol,nylidrin, diuretics, including furosemide, ethacrynic acid,spironolactone, triamterine, amiloride, thiazides, bumetanide, caffeine,theophylline, nicotine, captopril, salalasin, and potassium salts.

It has been found that elevated levels of homocysteine as a significantrisk in vascular disease, such as atherosclerosis, venous thrombosis,heart attack and stroke, as well as neural tube defects and neoplasia.Moghadasian et al., "Homocyst(e)ine and Coronary Artery Disease", Arch,Int. Med. 157(10):2299-2308 (Nov. 10, 1997), incorporated herein byreference. Homocystiene promotes free radical reactions. In patientswith defective homocysteine metabolism, relatively high levels ofhomocysteine are present in the blood. According to the presentinvention, glutathione is administered to patients with elevatedhomocysteine levels.

It was believed that, because hepatocytes produce reduced glutathionethrough a feedback-inhibited pathway, that hepatocytes would not absorbreduced glutathione from the plasma. Therefore, an insult tohepatocytes, for example from toxic or infectious origin, whichotherwise suppressed glutathione production would result in cellulardamage or death. In fact, the present inventors believe that this is notthe case, at least in the case of compromised hepatocytes. Therefore, itis an aspect according to the present invention to treat hepatitis, ofvarious types, with oral glutathione. For example, both alcohol andacetaminophen are both hepatotoxic, and result in reduced hepatocyteglutathione levels. Therefore, these toxicities may be treated accordingto the present invention. Glutathione may also be effective in thetreatment of other types of toxicities, to various cells or organs,which result in free radical damage to cells or reduced glutathionelevels.

Diabetes, especially uncontrolled diabetes, results in glycosylation ofvarious enzymes and proteins, which may impair their function orcontrol. In particular, the enzymes which produce reduced glutathione(e.g., glutathione reductase) become glycosylated and non-functional.Therefore, diabetes is associated with reduced glutathione levels, andin fact, many of the secondary symptoms of diabetes may be attributed toglutathione metabolism defects. The present invention may therefore beapplied to supplement diabetic patients with glutathione in order toprevent the major secondary pathology. The present invention alsoencompasses an oral pharmaceutical formulation comprising glutathioneand an antihyperglycemic agent.

Glutathione, due to its strong reducing potential, breaks disulfidebonds. It is believed that most normal proteins are not denatured, to agreat extent, by normal or superphysiologic levels of glutathione. It isbelieved, however, that opiate receptors are deactivated by high normallevels of glutathione. It is therefore believed that glutathioneadministration may be of benefit for the treatment of obesity and/oreating disorders, other addictive or compulsive disorders, includingtobacco (nicotine) and opiate additions.

The present invention also encompasses the administration of glutathionein conjunction with nicotine. The physiologic effects of nicotine arewell known. Glutathione, due to its vasodilatory effects, improvescerebral blood flow, resulting in a synergistic cerebralfunction-enhancing effect.

In mammals, the levels of glutathione in the plasma are relatively low,in the micromolar range, while intracellular levels are typically in themillimolar range. Therefore, the intracellular cytosol proteins aresubjected to vastly higher concentrations of glutathione thanextracellular proteins. The endoplasmic reticulum, a cellular organelle,is involved in processing proteins for export from the cell. It has beenfound that the endoplasmic reticulum forms a separate cellularcompartment from the cytosol, having a relatively oxidized state ascompared to the cytosol, and thereby promoting the formation ofdisulfide links in proteins, which are often necessary for normalactivity. Hwang, C., et al., "Oxidized Redox State of Glutathione in theEndoplasmic Reticulum", Science 257:1496-1502 (Sep. 11, 1992),incorporated herein be reference. In a number of pathological states,cells may be induced to produce proteins for export from the cells, andthe progression of the pathology interrupted by interference with theproduction and export of these proteins. For example, many viralinfections rely on cellular production of viral proteins forinfectivity. Interruption of the production of these proteins willinterfere with infectivity. Likewise, certain conditions involvespecific cell-surface receptors, which must be present and functional.In both these cases, cells which are induced to produce these proteinswill deplete reduced glutathione in the endoplasmic reticulum. It isnoted that cells which consume glutathione (GSH) will tend to absorbglutathione from the plasma, and may be limited by the amounts present.Therefore, by increasing plasma glutathione levels, even transiently,the reducing conditions in the endoplasmic reticulum may be interferedwith, and the protein production blocked. Normal cells may also besubjected to some interference; however, in viral infected cells, orcells abnormally stimulated, the normal regulatory mechanisms may not beintact, and the redox conditions in the endoplasmic reticulum controlledby the availability of extracellular glutathione. In these conditions,the pharmaceutical administration of glutathione may produce significanteffects.

Reproduction of herpes viruses, which are DNA viruses, is inhibited orreduced in cell culture by the administration of extracellularglutathione. Therefore, according to the present invention, herpes virusinfections may be treated by administering glutathione according to thepresent invention. The known herpes viruses include herpes simplex virusI, herpes simplex virus II, herpes zoster, cytomegalovirus, Epstein Barrvirus, as well as a number of other known viruses.

It is also believed that infection by the rabies virus, an RNA virus,may be treated by the administration of glutathione. While standardtreatments are available, and indeed effective when timely administered,glutathione may be useful in certain circumstances. Therefore, rabiesvirus infection may be treated, at least in part, according to thepresent invention. One available treatment for rabies is an immuneserum. The present invention therefore encompasses the parenteraladministration of glutathione in combination with an antibody.Glutathione may also be administered separately.

Coronary heart disease risk is increased by the consumption of ahigh-fat diet, and reduced by the intake of antioxidant vitamins,including vitamin E and vitamin C, as well as flavonoids. High fat mealsimpair the endothelial function through oxidative stress, resulting inimpaired nitric oxide availability. It has been found that vitamin C andvitamin E restores the vasoconstriction resulting from nitric oxideproduction by endothelium after a high fat meal. Plotnick, G. D. et al.,"Effect of Antioxidant Vitamins on the Transient Impairment ofEndothelium-Dependent Brachial Artery Vasoactivity Following a SingleHigh Fat Meal", JAMA 278:1682-1686 (Nov. 26, 1997), incorporated hereinby reference. According to the present invention, glutathione may beadministered as a prophylaxis against vascular disease.

In utilizing antioxidants as advanced therapeutic approaches, thefollowing principles have been developed over time:

Different disorders generate different types of free radicals, indifferent environments. Therefore, different specific antioxidants areneeded for these various radicals and related compounds. The commonestspecies and related molecules includes superoxide, •O₂ --; hydroxyl,•OH; peroxy, •OOH; hydrogen peroxide, H₂ O₂ (splitting into hydroxylradicals); alkoxy, RO•; delta singlet oxygen, ¹ O₂ ; nitric oxide, •NO;lipid hydroperoxides, LOOH (splitting into alkoxy and hydroxylradicals). See, Montaignier, Luc, Olivier, Rene, Pasquier,Catherine(Eds.), Oxidative Stress in Cancer, AIDS, and NeurodegenerativeDiseases, Marcel Dekker, NY (1998), incorporated herein by reference inits entirety.

In addition to qualitative differences among several species of freeradicals, their rates of formation will differ, as will the differenttypes of inciting agents that may have to be simultaneously controlled.For example, continued, unprotected exposures of the eyes, in MacularDegeneration, to strong sunlight and to tobacco smoke, would limitbenefits from an antioxidant used as a therapeutic agent for control ofthis disease. Therefore, one aspect of the invention providessynergistic therapies to patients by increasing antioxidant levelssystemically or in specific organs as well as reducing oxidative, freeradical generating and ionizing influences. In this case, glutathionetherapy would be complemented with ultraviolet blocking sunglasses, anda tobacco smoking cessation plan, if necessary. A particularlyadvantageous antioxidant for combination with glutathione is alphatocopherol succinate.

Free radicals occur in different parts or subparts of tissues and cells,with different inciting agents. For example, in trauma to the brain orspinal cord, the injurious free radicals are in the fatty (lipid)coverings that insulate nerve fibers, the myelin sheaths. Extremely highdoses of a synthetic corticosteroid, 5 to 10 grams of methylprednisolone sodium succinate (MPSS), given for just 24 hours, rapidlyreach the brain and spinal cord and diffuse rapidly into the myelin,neutralizing the trauma-induced radicals, specifically: •OH, •OOH, andRO•. It is therefore an object of the invention to provide apharmaceutical composition comprising a combination of glutathione and aglucocorticoid agent.

The accepted, published, peer-reviewed literature has repeatedlydemonstrated the multiple properties of glutathione in the body. Theabundant physiological and biochemical properties of glutathione ledothers into an extensive series of clinical trials wherein precursors ofglutathione were administered, because the prevailing belief was thatglutathione itself could not be effectively absorbed if it was simplygiven as glutathione. Hence, the popularity of the relativelyineffective and potentially damaging glutathione precursor N-acetylcysteine (NAC) is currently being misused in the homosexual (high AIDSrisk) community. The further belief was that glutathione would not crossthe membranes of lymphocytes and other cells, whereas NAC could. Theview was that to try to correct the glutathione deficiency in HIV/AIDS,with glutathione itself, was a hopeless task, because it would bedegraded before uptake across membranes. However, the precursors ofglutathione have failed to raise intracellular GSH levels. The presentinvention provides a suitable regimen to orally administer glutathioneto achieve high bioavailability and increased intracellular levels ofglutathione.

While prior studies have employed glutathione dissolved in orange juiceto administer glutathione to AIDS patients, resulting in glutathioneuptake, this method does not provide the advantages of an encapsulatedor pill form, and there was no recognition for the need to preventdigestive dilution or glutathione derived impurities from being present.

Glutathione has also proven to be an effective anti-viral agent andinterferes with HIV replication at a critical site that is not affectedby other current drugs, viral mRNA transcription. Glutathione keepsviral DNA quiescent, especially when potent activators are present, likeNFκB, and TNFα. Glutathione's anti-viral target appears to be at a pointwhere the virus can not readily mutate. The dependence of HIVreplication on binding activated NFκB onto its Long Terminal Repeat(LTR) appears to be central to the virus.

According to the present invention, orally administered glutathione cansafely raise cell levels beyond correcting glutathione deficiencies. Anumber of pathologic processes can be inhibited by these higher levels,for example, curtailing the virtually self-perpetuating, powerfulbiochemical cycles producing corrosive free radicals and toxic cytokinesthat are largely responsible for the signs and symptoms of AIDS. Thesebiochemical cycles destroy considerable quantities of glutathione butthey can eventually be brought under control, and normalized withsufficient, on-going glutathione therapy. A typical example is the overproduction of a substance, 15 HPETE (15-hydroperoxy eicosatetraenoicacid), from activated macrophages. The 15 HPETE is a destructive,immunosuppressing substance and requires glutathione for conversion intoa non-destructive, benign molecule. The problem is that once macrophagesare activated, they're difficult to normalize.

Once inside cells, GSH curtails the production of free radicals andcytokines, corrects the dysfunctions of lymphocytes and of macrophages,reinforces defender cells in the lungs and other organs, halts HIVreplication in all major infected cell types, by preventing theactivation of the viral DNA by precluding the activation of NFκB,inhibits the TAT gene product of HIV that drives viral replication,dismantles the gp120 proteins of the virus coat. These gp120 proteinsare the projections of the virus that normally allow it to lock ontosusceptible CD4+ cells thereby helping the spread of the virus touninfected CD4+ cells. By disrupting the gp120 protein, glutathioneoffers a potential mode of preventing transmission not only to othercells in the patient, but perhaps in precluding transmission to others.

Besides classic antiviral or antiretroviral agents (reversetranscriptase inhibitors, protease inhibitors), a number of othertherapies may be of benefit for AIDS patients, and the present inventionprovides combinations of glutathione with the following drugs:cyclosporin A, thalidomide, pentoxifylline, selenium, desferroxamine,2L-oxothiazolidine, 2L-oxothiazolidine-4-carboxylate,diethyldithiocarbamate (DDTC), BHA, nordihydroguairetic acid (NDGA),glucarate, EDTA, R-PIA, alpha-lipoic acid, quercetin, tannic acid,2'-hydroxychalcone, 2-hydroxychalcone, flavones, alpha-angelicalactone,fraxetin, curcurmin, probucol, and arcanut (areca catechul).

Inflammatory responses are accompanied by large oxidative bursts,resulting in large numbers of free radicals. Therefore, glutathione mayhave application in the therapy for inflammatory diseases. Glutathionemay advantageously reduce the primary insult a well as undesired aspectsof the secondary response. According to the present invention,glutathione may be administered to patients suffering from aninflammatory disease process, such as arthritis or various types,inflammatory bowel disease, etc. The present invention also providescombination pharmaceutical therapy including glutathione and ananalgesic or antiinflammatory agent, for example opiate agonists,glucocorticoids or non-steroidal antiinflammatory drugs (NSAIDS),including opium narcotics, meperidine, propoxyphene, nalbuphine,pentazocine, buprenorphine, asprin, indomethacin, diflunisal,acetominophen, ibuprofen, naproxen, fenoprofen, piroxicam, sulindac,tolmetin, meclofenamate, zomepirac, penicillamine, phenylbutazone,oxyphenbutazone, chloroquine, hydroxychloroquine, azathiaprine,cyclophosphamide, levamisole, prednisone, prednisolone, betamethasone,triamcinolone, and methylprednisolone.

Glutathione may also hold benefit for the treatment of parotitis,cervical dysplasia, Alzheimer's disease, Parkinson's disease,aminoquinoline toxicity, gentamycin toxicity, puromycin toxicity,aminoglycoside nephrotoxicity, paracetamol, acetaminophen and phenacetintoxicity,

Glutathione need not be orally ingested in order to provide thebeneficial effects noted. While the drug may be administeredintravenously or parenterally, it may also be administered throughmucous membranes, including sublingually, as a vaginal or rectalsuppository, and by pulmonary inhaler, for topical applications to thealveolar surface cells of the lungs to enhance pulmonary protectionagainst unusual pneumonias. Systemic administration of glutathione maybe used to concentrate glutathione in lymph nodes, and lymphoid tissues.

Glutathione tends to be unstable in solution. Therefore, one aspect ofthe present invention provides a pharmaceutical administration apparatusproviding a dual chamber distribution pouch, having a frangibleinterconnection, allowing mixing between an aqueous phase and a dryglutathione preparation. The aqueous phase may be, for example, a gel,cream or foam. Either pouch may also contain another pharmaceuticalagent, as described above.

The present invention also provides a glutathione administrationappliance, for delivering an effective dose of glutathione to anaccessible mucous membrane, such as the oral, vaginal, urethral or analcavities. A dry glutathione preparation, for example in a dehydratedgel, matrix or polymer, having a high surface area per unit volumeratio, is provided in a foil bag or pouch. The dehydrated mass includesglutathione, as well as an optional stabilizing agent, such as ascorbicacid. The dehydrated mass is hydrated by the mucosal membrane or by anexternally applied fluid, and the glutathione is then present to protectthe mucous membrane from viral infection.

The ability of glutathione to chemically dismantle the gp120 protein ofHIV by chemically destroying structural disulfide bonds, indicates thattransmission of the infection may be curtailed to some extent. If gp120is dismantled, the virus can not lock onto CD4+ cells. The oralglutathione treatment of patients may suffice to dismantle gp120 ofviruses from treated patients. The topical applications of glutathioneto mucous membranes might possibly serve to protect a sex partner ifunsafe sexual practices occur.

Another effect is seen when glutathione or nitroso-glutathione is placedin the male urethra. In this case, the glutathione or glutathionederivative is absorbed. The vasodilatory effects of nitroso-glutathione,which is formed by interaction of glutathione with nitric oxide orprovided directly, vasodilates the penis, resulting in an erection.Thus, a urethral glutathione or nitroso-glutathione suppository haspotential for the treatment of impotence.

Glutathione or a glutathione derivative may also be co-administered withyohimbine, an alpha-2 receptor blocker, providing a synergistic effect.Yohimbine has been established to treat male sexual dysfunction, (e.g.,impotence), among other effects.

Glutathione may be administered to mucous membranes in the form of aliquid, gel, cream, jelly, absorbed into a pad or sponge. Administrationmay also be provided by a powder or suspension.

The effective delivery of intact, pharmaceutically stabilized,bioavailable reduced L-glutathione has been accomplished according tothe present invention. By providing high-dose glutathione for the body'sgeneral use, diabetics having either form of the disease may be providedwith ample supplies of glutathione. Correcting the glutathionedeficiency and also raising the levels inside cells to the upper rangeof normal will help to delay, or prevent the complications of diabetes.

Glutathione, orally administered according to the present invention, inmoderately high doses, one to five gm/day, may be able to affect theoutcome of macular degeneration. The avidity with which the RPE cellstake up glutathione indicates that they may have a critical role inameliorating this disorder. Unlike rods and cones, RPE cells can divideand replenish themselves if allowed. If caught at an early stage, beforesignificant losses of rods and cones, the condition may be halted anddelayed possibly indefinitely.

Since glutathione is relatively non-toxic, it may be used liberally forits advantageous properties. According to one aspect of the invention,glutathione may be added to a viral contaminated fluid or potentiallycontaminated fluid to inactivate the virus. This occurs, for example, byreduction of critical viral proteins. According to a preferredembodiment, glutathione is added to blood or blood components prior totransfusion. The added glutathione is in the reduced form, and is addedin a concentration of between about 100 micromolar to about 500millimolar or to a solubility limit, whichever is lower, and morepreferably in a concentration of about 10-50 millimolar.

The addition of glutathione to whole blood, packed red blood cells orother formed blood components (white blood cells, platelets) may be usedto increase the shelf life and/or quality of the cells or formedcomponents.

EXAMPLE 1

Reduced L-glutathione, a naturally-occurring water-soluble tripeptide(gamma-glutamyl-cysteinyl-glycine) is the most prevalent intracellularthiol in most biological systems. A preferred formulation of glutathioneaccording to the present invention provides capsules for oral usecontaining 500 mg reduced L-glutathione, 250 mg USP grade crystallineascorbic acid, and not more than 0.9 mg magnesium stearate, NF grade inan OO-type gelatin capsule.

EXAMPLE 2

The preferred regimen for treatment of humans with glutathione accordingto the present invention is the administration of between 1 and threegrams per day, in two divided doses, between meals (on an emptystomach), of encapsulated, stabilized glutathione according toExample 1. The study detailed in Appendix B administered the glutathioneto HIV infected, otherwise healthy males between 18 and 65, with CD4+cell counts above 500, not on any other medications. As detailed in FIG.1, clinical responses were seen in the PBM intracellular glutathionelevels. Thus, at 1 hour after administration of a 1 gram bolus ofencapsulated stabilized glutathione in two 500 mg capsules, a three-foldincrease in glutathione was measured. It is noted that, since the humanbody produces large quantities of glutathione, the effects of externalglutathione in individual cases may sometimes be masked or even appearparadoxical. However, as shown in FIG. 2, a statistical analysis shows adose response effect of the administration of glutathione according tothe present invention to the subject population.

EXAMPLE 3

FIG. 2 shows a graph from one of the 24 HIV positive people in theCompany's Clinical Trial. The graph illustrates increases in theglutathione (GSH) content of immune system cells, in the blood,resulting from two doses of pharmaceutically stabilized GSH according toExample 1. The first dose of one gram was taken at 0 time, or 10:00 a.m.and the second dose at 3 hours, or 1:00 p.m. The baseline points werefrom two weeks earlier, on the same patient. A temporary intravenouscatheter was in place for 7 hours to permit frequent blood sampling atthe numerous time points. The units are in nanomoles of GSH per 10million peripheral blood mononuclear cells (PBMC's). The graph is anexample of the elevation of GSH inside PBMC's. The statistical analysisof the entire patient population shows statistically significantelevations and a significant dose response relationship.

In a compressed Phase I/II clinical trial (FDA IND#45012), in a welldefined GSH deficiency state, HIV infection, the composition accordingto Example 1 administered according to the protocol of Example 2 wasdemonstrated to rapidly and safely raises intracellular GSH levels twoto three fold. Thus, by employing the composition according to Example 1administered according to the protocol of Example 2, an oralpharmaceutical has been shown to treat the critical losses of GSH thatare known to propel a range of major disorders.

The glutathione metabolism, especially the pharmacokinetics, of thesubjects of the Phase II study is believed to be relatively normal.Therefore, the same regimen may be applied in the treatment of otherconditions, including CHF, diabetes, early stroke or other ischemicevent, toxic insult, viral infection or disease, or other condition inwhich free radical reactions are uncontrolled, aberrant, or contributeto pathology.

EXAMPLE 4 Combination of Glutathione and Acetaminophen

A combination pharmaceutical is provided to ameliorate the detrimentaleffects of acetaminophen, a drug which consumes glutathione in the liverduring metabolism, and in excess doses causes liver damage due tooxidative damage. The composition includes 500 mg L-glutathione, 250 mgcrystalline ascorbic acid, and 350 mg acetaminophen.

EXAMPLE 5 Combination of Glutathione and Chlorpromazine

A combination pharmaceutical is provided to ameliorate the detrimentaleffects of chlorpromazine, a phenothiazine drug which causes sideeffects, including tardive dyskinesia, possibly relating to excess freeradical reactions. The composition includes 500 mg L-glutathione, 250 mgcrystalline ascorbic acid, and 200 mg chlorpromazine.

EXAMPLE 6 Combination of Glutathione and Aminoglycosides

A combination pharmaceutical is provided to ameliorate the detrimentaleffects of Aminoglycoside drugs, which include, but are not limited to,neomycin, kanamycin, amikacin, streptomycin, gentamycin, sisomicin,netilmicin and tobramycin, a drug class which may be associated withvarious toxicities. This damage may be related to oxidative damage orconsumption of glutathione during metabolism. The composition accordingto the present invention is an intravenous formulation, including theaminoglycoside in an effective amount, and L-glutathione in an amount ofabout 10-20 mg/kg. Ascorbic acid in an amount of 5-10 mg/kg may be addedas a stabilizer.

EXAMPLE 7 Urethral Insert

A composition containing 200 mg glutathione, 50 mg ascorbic acid perunit dosage is mixed with carageenan and/or agarose and water in aquick-gelling composition, and permitted to gel in a cylindrical formhaving a diameter of about 3 mm and a length of about 30 mm. Thecomposition is then subjected to nitric oxide to cause between 0.1-10%of the glutathione to be converted to nitroso-glutathione. The gelledagarose is then freeze dried under conditions which allow shrinkage. Thefreeze dried gel is than packaged in a gas barrier package, such as afoil pouch or foil "bubble-pack".

The freeze dried gel may then be used as a source of nitroso-glutathionefor administration transmucosally. The cylindrical freeze dried gel maybe inserted into the male urethra for treatment of impotence, oradministered sublingually for systemic vasodilation.

EXAMPLE 7 Vascular Disease Prophylaxis

An oral formulation is provided for prophylaxis of vascular disease,e.g., in men over 40. The composition includes 500 mg reducedL-glutathione, 250 mg USP grade crystalline ascorbic acid, and 50 mg USPacetyl salicylic acid (aspirin) in an OO-type gelatin capsule. Typicaladministration is twice per day.

Advantageously, the acetyl salicylic acid may provided in entericrelease pellets within the capsule, slowing release.

EXAMPLE 8 Vascular Disease Prophylaxis

Arginine is the normal starting substrate for the production of nitricoxide. Arginine is normally in limited supply, and thus a relativedeficiency of arginine may result in impaired vascular endothelialfunction.

An oral formulation is provided for prophylaxis of vascular disease. Thecomposition includes 500 mg reduced L-glutathione, 200 mg USP gradecrystalline ascorbic acid, and 200 mg arginine, in an OO-type gelatincapsule.

EXAMPLE 9 Vascular disease Prophylaxis

Vitamin E consumption reduces the risk of heart attack and othervascular disease. Vitamin E succinate (alpha-tocopherol succinate) is adry powder.

An oral formulation is provided for prophylaxis of vascular disease. Thecomposition includes 500 mg reduced L-glutathione, 200 mg USP gradecrystalline ascorbic acid, and 200 mg vitamin E succinate, in an OO-typegelatin capsule.

EXAMPLE 10 Vascular Disease Prophylaxis

Nonspecific esterases are present in the plasma which have a broadsubstrate specificity. According to the present invention, esters areformed between agents which are useful combination therapies, in orderto provide for efficient administration, high bioavailability, andpharmaceutical stability. Preferred esters include alphatocopherol-ascorbate, alpha tocopherol-salicylate, andascorbyl-salicylate. The tocopherol ester maintains the molecule in areduced state, allowing full antioxidant potential after ester cleavage.

These esters may be administered alone or in combination with otheragents, for example glutathione. Typically, these are administered todeliver an effective dose of salicylate equivalent of 100 mg per day forprophylaxis or 750-1000 mg per dose for treatment of inflammatorydiseases. Tocopherol is administered in an amount of 100-500 IUequivalent. Ascorbate is administered in an amount of up to 1000 mgequivalent.

In order to enhance availability, a non-specific esterase may beprovided in the formulation to cleave the ester after dissolution of thecapsule. Therefore, a non-specific esterase, such as a bacterial orsaccharomyces (yeast) enzyme or enriched enzyme preparation may beincluded in the formulation, such as included as a powder or as pelletsin the capsule.

EXAMPLE 11 Vascular Disease Prophylaxis

Nordihydroguaretic acid is a known lipoxygenase inhibitor. Thiscomposition may therefore be used to treat inflammatory processes or asprophylaxis against vascular disease.

An oral formulation is provided for prophylaxis of vascular disease. Thecomposition includes 500 mg reduced L-glutathione, 200 mg USP gradecrystalline ascorbic acid, and 100 mg nordihydroguaretic acid, in anOO-type gelatin capsule. Typical administration is twice per day.

It should be understood that the preferred embodiments and examplesdescribed herein are for illustrative purposes only and are not to beconstrued as limiting the scope of the present invention, which isproperly delineated only in the appended claims.

REFERENCES

Each of the following references is incorporated herein in its entirety:

Glutathione, General.

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Aruga, M., Awazu, S. and Hanano, M.: Kinetic studies on decomposition ofglutathione. II. Anaerobic decomposition in aqueous solution. Chem.Pharm. Bull. 28:514-20, 1980.

Aruga, M., Awazu, S. and Hanano, M.: Kinetic studies on decomposition ofglutathione. III. Peptide bond cleavage and desulfurization in aqueoussolution. Chem. Pharm. Bull. 28:521-28, 1980.

Hagen, T. M., Aw, T. Y., and Jones, D. P.: Glutathione uptake andprotection against oxidative injury in isolated kidney cells. KidneyIntl. 34:74-81, 1988.

Lash. L. H., and Jones, D. P.: Distribution of oxidized and reducedforms of glutathione and cysteine in rat plasma. Arch. Biochem. Biophys.240:583-92, 1985.

Meister, A.: Selective modification of glutathione metabolism. Science220:472-477, 1983.

Meister, A. and Anderson, M. E.: Glutathione. Ann. Rev. Biochem.52:711-60, 1983.

Riley, R. J., Spielberg, S. P., Leeder, J. S.: A comparative study ofthe toxicity of chemically reactive xenobiotics towards adherent cellcultures: selective attenuation of menadione toxicity by buthioninesulphoximine pretreatment. J. Pharmacol. 45(4): 263-267, 1993.

Wierzbicka, G. T., Hagen, T. M. & Jones, D. P.: Glutathione in food. J.Food Comp. Anal. 2:327-337, 1989.

Glutathione and the Immune System.

Droge, W., Pottmeyer-Gerber, C., Schmidt, H. & Nick, S.: Glutathioneaugments the activation of cytotoxic T lymphocytes in vivo. Immunobiol.172:151-156, 1986.

Droge, W., Eck, H. P., Gmunder, H., and Mihm, S.: Modulation oflymphocyte functions and immune responses by cysteine and cysteinederivatives. Amer. J. Medicine 91(3C):140S-144S, 1991.

Furukawa, T., Meydani, S. N. & Blumberg, J. B.: Reversal ofage-associated decline in immune responsiveness by dietary glutathionesupplementation in mice. Mech. Ageing Dev. 38:107-117, 1987.

Franklin, R. A., Yong, M. L., Arkins, S., and Kelley, K. W.: Glutathioneaugments in vitro proliferative responses of lymphocytes to concanavalinA to a greater degree in old than in young rats. J. Nutr. 120:1710-17,1990.

Kavanaugh, T. J., Grossman, A., Jaecks, E. P, Jinneman, J. C., Eaton, D.L., Masrtin, G. M., and Rabinovitch, P. S.: Proliferative capacity ofhuman peripheral lymphocytes sorted on the basis of glutathione content.J. Cell. Physiol. 145:472-80, 1990.

Robinson, M. K, Rodrick, M. L., Jacobs, D. O., Rounds, J. D., Collins,K. H., Saproschetz, I. B., Mannick, J. A., and Wilmore, D. W.:Glutathione depletion in rats impairs T-cell and macrophage immunefunction. Arch. Surg. 128:29-35, 1993.

Suthanthiran, M., Anderson, M. E., Sharma, V. K. & Meister, A.:Glutathione regulates activation-dependent DNA synthesis in highlypurified normal human T lymphocytes stimulated via the CD2 and CD3antigens. Proc. Natl. Acad. Sci. USA 87:3343-3347, 1990.

Glutathione as a Detoxicant.

Bravenboer, B., Kappelle, A. C., Hamers, F. P., van Buren, T., Erkelens,D. W. & Gispen, W. H.: Potential use of glutathione for the preventionand treatment of diabetic neuropathy in the streptozocin-induceddiabetic rat. Diabetologia 35:813-817, 1992.

Cavaletti, E., Tofanetti, O. & Zunino F.: Comparison of reducedglutathione with 2-mercaptoethane sulfonate to preventcyclophosphamide-induced urotoxicity. Cancer Letters 32:1, 1986.

Hamers, F. P., Brakkee, J. H., Cavalletti, E., Tedeschi, M., Marmonti,L., Pezzoni, G., Neijt, J. P. & Gispen, W. H.: Reduced glutathioneprotects against cisplatin-induced neurotoxicity in rats. Cancer Res.53:544-549, 1993.

Kromidas, L., Trombetta, L. D., and Jamall, I. S.: The protectiveeffects of glutathione against methylmercury cytotoxicity. Toxicol.Letters 51:67-80, 1990.

Novi, A. M., Flohe, R., and Stukenkemper, S.: Glutathione and aflatoxinB₁ -induced liver tumors: requirement for an intact glutathione moleculefor regression of malignancy in neoplastic tissue. Ann. NY Acad. Sci.397:62-71, 1982.

Rao, R. D. N., Fischer, V., and Mason, R. P.: Glutathione and ascorbatereduction of the acetaminophen radical formed by peroxidase. J. Biol.Chem. 265:844-7, 1990.

Skoulis, N. P., James, R. C., Harbison, R. D. and Roberts, S. M.:Depression of hepatic glutathione by opioid analgesic drugs in mice.Toxicol. Appl. Pharmacol. 99:139-47, 1989.

Villani, F., Galimberti, M., Zunino, F., Monti, E., Rozza, A., Favalli,L. & Poggi, P.: Prevention of doxorubicin-induced cardiomyopathy byreduced glutathione. Cancer Chemother. Pharmacol. 28:365-369, 1991.

Wagner, G., Frenzel, H., Wefers, H. and Sies, H.: Lack of effect oflong-term glutathione administration on aflatoxin B1-induced hepatoma inmale rats. Chem. Biol. Interactions 53:57-68, 1985.

Yoda, Y., Nakazawa, M., Abe, T. & Kawakami, Z.: Prevention ofDoxorubicin myocardial toxicity in mice by reduced glutathione. CancerResearch 46:2551, 1986.

Younes, M., and Strubelt, O.: Protection by exogenous glutathioneagainst hypoxic and cyanide-induced damage to isolated perfused ratlivers. Toxicol. Letters 50:229-236, 1990.

McCartney, M. A.: Effect of glutathione depletion on morphine toxicityin mice. Biochem. Pharmacol. 38:207-9, 1989.

Ishida, T., Kumagai, Y., Ikeda, Y., Ito, K., Yano, M., Toki, S.,Mihashi, K., Fujioka, T., Iwase, Y. and Hachiyama, S.:(8S)-(glutathion-S-YL)dihydromorphinone, a novel metabolite kof morphinefrom guinea pig bile. Drug. Metab. Dispos. 17:77-81, 1989.

Nagamatsu, K., Kido, Y., Teroa, T, Ishida, T. and Toki, S.: Protectiveeffect of sulfhydryl compounds on acute toxicity of morphinone. LifeSci. 30:1121-27, 1982.

Glutathione as an Adjunct to Cancer Chemotherapy.

Bohm, S., Battista-Spatti, G., DiRe, F., Oriana, S., Pilotti, S.,Tedeschi, M., Tognella, S. & Zunino, F.: A feasibility study ofcisplatin administration with low-volume hydration and glutathioneprotection in the treatment of ovarian carcinoma. Anticancer Res.11:1613-1616, 1991.

Cozzaglio, L., Doci, R., Colla, G., Zunino, F., Casciarri, G. & Gennari,L.: A feasibility study of high-dose cisplatin and 5-fluorouracil withglutathione protection in the treatment of advanced colorectal cancer.Tumori 76:590-594, 1990.

Di Re, F., Bohm, S., Oriana, S., Spatti, G. B., & Zunino, F.: Efficacyand safety of high-dose cisplatin and cyclophosphamide with glutathioneprotection in the treatment of bulky advanced epithelial ovarian cancer.Cancer Chemother. Pharmacol. 25:355-360, 1990.

Nobile, M. T., Vidili, M. G., Benasso, M., Venturini, M., Tedeschi, M.,Zunino, F., & Rosso, R.: A preliminary clinical study ofcyclophosphamide with reduced glutathione as uroprotector. Tumori75:257-258, 1989.

Glutathione Use in Patients with Abnormal Glucose Tolerance, and withDiabetes.

Ceriello, A., Giugliano, D., Quatraro, A. & Lefebvre, P. J.:Anti-oxidants show an anti-hypertensive effect in diabetic andhypertensive subjects. Clin. Sci. 81:739-742, 1991.

Paolisso, G., Giugliano, D., Pizza, G., Gambardella, A., Tesauro, P.,Varricchio, M. & D'Onofrio, F.: Glutathione infusion potentiatesglucose-induced insulin secretion in aged patients with impaired glucosetolerance. Diabetes Care 15:1-7, 1992.

Glutathione as a Treatment for Renal Failure.

Costagliola, C., Romano, L., Scibelli, G., de Vincentiis, A., Sorice, P.& DiBenidetto, A.: Anemia and chronic renal failure: a therapeuticapproach by reduced glutathione parenteral administration. Nephron61:404-408, 1992.

Toxicological Effects of Glutathione.

Dalhoff, K., Ranek, L., Mantoni, M. & Poulsen, H. E.: Glutathionetreatment of hepatocellular carcinoma. Liver 12:341-343, 1992.

Dekant, W.: Bioactivation of nephrotoxins and renal carcinogens byglutathione S-conjugate formation. Toxicol. Letters 67:151-60, 1993.

Domingo, J. L., Gomez, M., Llobet, J. M. & Corbella, J.: Chelatingagents in the treatment of acute vanadyl sulphate intoxication in mice.Toxicology 62: 203-211, 1990.

Martensson, J., Han, J., Griffith, O. W. & Meister, A.: Glutathioneester delays the onset of scurvy in ascorbate-deficient guinea pigs.Proc. Nat. Acad. Sci. USA 90:317-321, 1993.

Thust, R, and Bach, B.: Exogenous glutathione induces sister chromatidexchanges, clastogenicity and endoreduplication in V79-E Chinese hamstercells. Cell Biol. Toxicol. 1:123-31, 1985.

HIV Infection.

Arpadi, S. M., Zang, E, Muscat J. and Richie, J.: Glutathione deficiencyin HIV-1-infected children with growth failure, (submitted forpublication).

Baker, D. H. and Wood, R. J.: Cellular antioxidant status and humanimmunodeficiency virus replication. Nutr. Rev. 50:15-8, 1992.

Baruchel, S., and Wainberg, M. A.: The role of oxidative stress indisease progression in individuals infected by the humanimmunodeficiency virus. J. Leukocyte Biol. 52:111-114, 1992.

Buhl, R., Holroyd, K. J., Mastrangli, A., Cantin, A. M., Jaffe, H. A.,Wells, F. B., Saltini, C. and Crystal, R. G.: Systemic glutathionedeficiency in symptom-free HIV-seropositive individuals. Lancetii:1294-1298, 1989.

de Quay, B., Malinverni, R. and Lauterburg, B. H.: Glutathione depletionin HIV-infected patients: role of cysteine deficiency and effect of oralN-acetylcysteine. AIDS 6:815-9, 1992.

Droge, W., Eck, H. P. and Mihm, S.: HIV-induced cysteine deficiency andT-cell dysfunction--a rationale for treatment with N-acetylcysteine.Immunol. Today 13:211-4, 1992.

Eck, H. P., Gmunder, H., Hartmann, M., Petzoldt, D., Daniel, V. andDroge, W.: Low concentrations of acid-soluble thiol (cysteine) in theblood plasma of HIV-1-infected patients. Biol. Chem. Hoppe-Seyler 370:101-108, 1989.

Fauci, A. S.: Multifactorial nature of human immunodeficiency virusdisease: Implications for therapy. Science 262:1011-1018, 1993.

Foley, P. Kazazi, F., Biti, R., Sorrell, T. C., and Cunningham, A. L.:HIV infection of monocytes inhibits the T-lymphocyte proliferativeresponse to recall antigens via production of eicosanoids. Immunology75:391-97, 1992.

Hasan, V., Thomas, D., Aclami, J. et al.: Stimulation of a human T-cellclone with anti-CD3 or tumor necrosis factor induces NFkB translocationbut not human immunodeficiency virus 1 enhancer-dependent transcription.Proc. Natl. ACAD. sCI. 87:7861-65, 1990.

Ho, W. Z. and Douglas, S. D.: Glutathione and N-acetylcysteinesuppression of human immunodeficiency virus replication in humanmonocyte/macrophages in vitro. AIDS Res. Hum. Retroviruses, 8:1249-53,1992.

Israel, N., Gougerot-Pocidalo, M. A., Aillet, F., and Virelizier, J. L.:Redox status of cells influences constitutive or induced NFκBtranslocation and HIV long terminal repeat activity in human T andmonocytic cell lines. J. Immunol. 149:3386-93, 1992.

Kobayashi, S., Hamamoto, Y., Kobayashi, N., and Yamamoto, N.: Serumlevel of TNFa in HIV-infected individuals. AIDS 4:169 1990.

Kalebic, T., Kinter, A., Poli, G., Anderson, M. E., Meister, A. andFauci, A. S.: Suppression of human immunodeficiency virus expression inchronically infected monocytic cells by glutathione, glutathione ester,and N-acetylcysteine. Proc. Natl. Acad. Sci. USA 87: 986-990, 1991.

LeGrand-Poels, S., Vaira, D., Pincemail, J., Van de Vorst, A. andPiette, J.: Activation of human immunodeficiency virus type 1 byoxidative stress. AIDS Res. Hum. Retrov. 6:1389-97, 1990.

Mihm, S., Ennen, J., Pessara, U., Kurth, R. and Droge, W.: Inhibition ofHIV-1 replication and NF-kb activity by cysteine and cysteinederivatives. AIDS 5:497-503, 1991.

National Institutes of Health. Dr. Howard C. Greenspan, Chairman ofConference on Free Radicals and Antioxidants in HIV/AIDS, Nov. 12-13,1993/Greenspan, H. C. The role of reactive oxygen species, antioxidantsand phytopharmaceuticals in human immunodeficiency virus activity.Med-Hypotheses 40: 85-92, 1993.

Roederer, M., Raju, P. A., Staal, F. J. T., Herzenberg, L. A. andHerzenberg, L. A.: N-acetylcysteine inhibits latent HIV expression inchronically infected cells. AIDS Res. Human Retrovir. 7:(6) 563-567,1991.

Roederer, M., Staal, F. J. T., Osada, H., Herzenberg, L. A. andHerzenberg, L. A.: CD4 and CD8 T cells with high intracellularglutathione levels are selectively lost as the HIV infection progresses.Internat. Immunol. 3:933-37, 1991.

Roederer, M., Staal, F. J. T., Raju, P. A., Ela, S. W., Herzenberg, L.A. and Herzenberg, L. A.: Cytokine-stimulated human immunodeficiencyvirus replication is inhibited by N-acetyl-L-cysteine. Proc. Natl. Acad.Sci. USA 87: 4884-4888, 1990.

Schreck, R. Rieber, P., and Baeurle, P. A.: Reactive oxygenintermediates as apparently widely used messengers in the activation ofthe NF-kb transcription factor and HIV-1. EMBO J. 10:2247-2258, 1991.

Staal, F. J., Roederer, M., Herzenberg, L. A. and Herzenberg, L. A.:Glutathione and immunophenotypes of T and B lymphocytes in HIV-infectedindividuals. Ann. NY Acad. Sci. 651:453-63, 1992.

Staal, F. J. T., Roederer, M. Herzenberg, L. A., and Herzenberg, L. A.:Intracellular thiols regulate activation of nuclear factor kappa-B andtranscription of human immunodeficiency virus. Proc. Natl. Acad. Sci.USA 87: 9943-9947, 1990.

Staal, F. J., Ela, S. W., Roederer, M., Anderson, M. T., Herzenberg, L.A. and Herzenberg, L. A.: Glutathione deficiency and humanimmunodeficiency virus infection. Lancet 339:909-12, 1992.

Staal, F. J., Roederer, M., Israelski, D. M., Bubp, J., Mole, L. A.,McShane, D., Deresinski, S. C., Ross, W., Sussman, H., Raju, P. A.,Herzenberg, L. A. and Herzenberg, L. A.: Intracellular glutathionelevels in T cell subsets decrease in HIV-infected individuals. AIDS Res.Hum. Retroviruses 8:305-11, 1992.

Staal, F. J. T., Roederer, M., Raju, P. A., Anderson, M. T., Ela, S. W.,Herzenberg, L. A., and Herzenberg, L. A.: Antioxidants inhibitstimulation of HIV transcription. AIDS Res. Hum. Retrov. 9:299-306,1993.

Wahl, L. M., Corcoran, M. L., Pyle, S. W., Arthur, L. O., Harel-Bellan,A. and Farrar, W. L.: Human immunodeficiency virus glycoprotein (gp120)induction of monocyte arachidonic acid metabolites and interleukin 1.Proc. Natl. Acad. Sci. 86:621-625, 1989.

Measurement of Glutathione.

Fahey, R. C., and Newton, G. L.: Determination of low molecular weightthiols using monobromobimane fluorescent labeling and high-performanceliquid chromatography. Meth. Enzymol. 143:85-96, 1987.

Mills, B. J., Richie. J. P. Jr., and Lang, C. A.: Sample processingalters glutathione and cysteine values in blood. Anal. Biochem.184:263-267, 1990.

Richie, J. P. Jr., and Lang. C. A.: The determination of glutathione,cyst(e)ine, and other thiols and disulfides in biological samples usinghigh-performance liquid chromatography with dual electrochemicaldetection. Anal. Biochem. 163:9-15, 1987.

Tietz, F.: Enzymic method for quantitative determination of nanogramamounts of total and oxidized glutathione: Applications to mammalianblood and other tissues. Anal. Biochem. 27:502-22, 1969.

Pharmacokinetics and Biological Disposition of Glutathione in Animals.

Aebi, S. & Lauterberg, B. H.: Divergent effects of intravenous GSH andcysteine on renal and hepatic GSH. Aer. J. Physiol. 263(2 pt2):R348-R352, 1992.

Ammon, H. P. T., Melien, M. C .M. & Verspohl, E. J.: Pharmacokinetics ofintravenously administered glutathione in the rat. J. Pharm. Pharmacol.38:721-725, 1986.

Anderson, M. E., Powrie, F., Puri, R. N., & Meister, A.: Glutathionemonoethyl ester: Preparation, uptake by tissues, and conversion toglutathione. Arch. Biochem. Biophys. 239:538-548, 1985.

Aw, T. Y., Wierzbicka, G. & Jones, D. P.: Oral glutathione increasestissue glutathione in vivo. Chem. Biol. Interact. 80:89-97, 1991.

Borok, Z., Buhl, R., Grimes, G. J., Bokser, A. D., Hubbard, R. C.,Holroyd, K. J., Roum, J. H., Czerski, D. B., Cantin, A. M., & Crystal,R. G.: Effect of glutathione aerosol on oxidant-antioxidant imbalance inidiopathic pulmonary fibrosis. The Lancet 338:215-216, 1991.

Buhl, R., Vogelmeier, C., Critenden, M., Hubbard, R. C., Hoyt, Jr., R.F., Wilson, E. M., Cantin, A. M. & Crystal, R. G.: Augmentation ofglutathione in the fluid lining the epithelium of the lower respiratorytract by directly administering glutathione aerosol. Proc. Natl. Acad.Sci. USA 87:4063-4067, 1990.

Bump, E. A., al-Sarraf, R., Pierce, S. M. & Coleman, C. N.: Elevation ofmouse kidney thiol content following administration of glutathione.Radiother. Oncol. 23:21-25, 1992.

Griffith, O. W., Bridges, R. J., & Meister, A.: Formation ofg-glutamyl-cyst(e)ine in vivo is catalyzed by g-glutamyl transpeptidase.Proc. Natl. Acad. Sci. USA 78:2777-2781, 1981.

Hagen, T. M., Wierzbicka, G. T., Bowman, B. B., Aw, T. Y. & Jones, D.P.: Fate of dietary glutathione. Disposition in the gastrointestinaltract. Am. J. Physiol. 259: G530-G535, 1990.

Hagen, T. M. & Jones, D. P.: Transepithelial transport of glutathione invascularly perfused small intestine of rat. Am. J. Physiol.252:G607-G613, 1987.

Hagen, T. M., Wierzbicka, G. T., Sillau, A. H., Bowman, B. B. & Jones,D. P.: Bioavailability of dietary glutathione. Effect on plasmaconcentration. Am. J. Physiol. 0 259:G524-G529, 1990.

Hahn, R., Wendel, A. & Flohe, L.: The fate of extracellular glutathionein the rat. Biochim. Biophys. Acta 539:324-337, 1978.

Puri, R. N., & Meister, A.: Transport of glutathione, asg-glutamylcysteinylglycyl ester, into liver and kidney. Proc. Natl.Acad. Sci. USA 80:5258-5260, 1983.

Vina, J., Perez, C., Furukawa, T., Palacin, M. & Vina, J. R.: Effect oforal glutathione on hepatic glutathione levels in rats and mice. Brit.J. Nutr. 62:683-91, 1989.

Pharmacokinetics of Glutathione in Humans.

Aebi, S., Asserto, R., & Lauterberg, B. H.: High-dose intravenousglutathione in man.: Pharmacokinetics and effects on cyst(e)ine levelsin plasma and urine. Eur. J. Clin. Invest. 21:103-110,1991.

Hagen, T. M. and Jones, D. P. Role of glutathione transport inextrahepatic detoxication. in Glutathione Centennial: MolecularPerspectives and Clinical Implications, N. Taniguchi, T. Higashi, Y.Sakamoto and A. Meister, eds. Acad. Press, New York, 1990.

Jones, D. P., Hagen, T. M., Weber, R., Wierzbicka, G. T., and Bonkovsky,H. L.: Oral administration of glutathione (GSH) increases plasma GSHconcentration in humans. FASEB J. 3:A1250 (5953), 1990.

Inflammation

Kuehl, F. A., Ham, E. A., Egan, R. W., Dougherty, H. W., Bonney, R. J.and Humes, J. L.: Studies on a destructive oxidant released in theenzymatic reduction of prostaglandin G2 and other hydroperoxy acids. In:Pathology of Oxygen, ed. A. P. Auton, Acad. Press, New York, 1982, pp.175-190.

Lash, L. H., Hagen, T. M., & Jones, D. P.: Exogenous glutathioneprotects intestinal epithelial cells from oxidative injury. Proc. Natl.Acad. Sci. USA 83:4641-4645, 1986.

Vascular effects of Glutathione

Demopoulos, H. B., Flamm, E. S., Pietronigro, D. D., and Seligman, M.L.: Free radical pathology and antioxidants in regional cerebralischemia and central nervous system trauma. In: Anesthesia andNeurosurgery, eds. J. E. Cottrell and H. Tunndorf C. V. Mosby, St.Louis, 1986, pp. 246-279.

Kagan, V. E., Bakalova, R. A., Koynova, G. M., Tyurin, V. A., Seriniva,E. A., Petkov, V. V., Staneva, D. S. and Packer, L.: Antioxidantprotection of the brain against oxidative stress. In: Free Radicals inthe Brain., eds. L. Packer, L. Prilipko, and Y. Christen.Springer-Verlag, New York, 1992, pp. 49-61.

Pietronigro, D. D., Demopoulos, H. B., Hovsepian, M. and Flamm, E. S.:Brain ascorbic acid depletion during cerebral ischemia. Stroke13:117-119, 1982.

Shan, X., Aw, T. Y. and Jones, D. P.: Glutathione-dependent protectionagainst oxidative injury. Pharmac. Ther. 47:61-71, 1990.

Simon, D. I., Stamler, J. S., Jaraki, O., et al.: Antiplateletproperties of protein S-nitrosothiols derived from nitric oxide andendothelium-derived relaxing factor. Arterioscler. Thromb.13(6):791-799, 1993.

Taccone-Gallucci, M., Lubrano, R., Clerico, A., Meloni, C., Morosetti,M., Meschini, L., Elli, M., Trapasso, E., Castello, M. A. & Casciani, C.U.: Administration of GSH has no influence on the RBC membrane:Oxidative damage to patients on hemodialysis. ASAIO Journal 38:855-857,1992.

Miscellaneous

Lenzi, A., Lombardo, F., Gandini, L., Culasso, F. & Dondero, F.:Glutathione therapy for male infertility. Arch. Androl. 29:65-68, 1992.

What is claimed is:
 1. A method of increasing glutathione levels inmammalian cells comprising administering an oral bolus of encapsulatedglutathione, pharmaceutically stabilized with an acidic reducing agent,in a rapidly dissolving formulation to a mammal on an empty stomach. 2.The method of claim 1 wherein the mammal is infected with an RNA virus.3. The method according to claim 2 wherein the virus is a retrovirus. 4.The method according to claim 3 wherein the retrovirus is HIV.
 5. Themethod according to claim 4 wherein the mammal is a human having a CD4+cell count of greater than
 500. 6. The method according to claim 4wherein the mammal is a human having AIDS.
 7. The method according toclaim 2 wherein the virus is the rabies virus.
 8. The method accordingto claim 1 wherein the mammal is infected with a herpes virus.
 9. Themethod according to claim 8 wherein the herpes virus is a human herpesvirus.
 10. The method according to claim 1, wherein the mammal hasdiabetes.
 11. The method according to claim 1 wherein the mammal haslevels of blood glucose which are physiologically elevated.
 12. Themethod according to claim 11 wherein enzymes in the mammalian cellswhich produce reduced glutathione are glycated.
 13. The method accordingto claim 1, wherein the mammal has congestive heart failure.
 14. Themethod according to claim 1, wherein the mammal has vasoconstrictionresulting from poor utilization of nitric oxide and resulting increasedperipheral vascular resistance.
 15. The method according to claim 1wherein the mammal is exposed to a toxic compound which promoteduncontrolled free radical oxidation.
 16. The method according to claim15, wherein the compound is an alcohol.
 17. The method according toclaim 16, wherein the alcohol is ethanol.
 18. The method according toclaim 15, wherein the compound is acetaminophen.
 19. The methodaccording to claim 1 wherein the mammal has hepatitis.
 20. The methodaccording to claim 19 wherein the hepatitis is an infectious hepatitis.21. The method according to claim 1 wherein the encapsulatedpharmaceutically stabilized glutathione in a rapidly dissolvingformulation comprises about 500 mg of glutathione and about 250 mg ofcrystalline ascorbic acid in a hard gelatin capsule.
 22. The methodaccording to claim 1, wherein the glutathione is pharmaceuticallystabilized with ascorbic acid.
 23. The method according to claim 22,wherein the ascorbic acid is present in an amount of about 1:1 to 1:10to glutathione by weight.
 24. The method according to claim I whereinthe glutathione is encapsulated with an antistatic agent.
 25. The methodaccording to claim 24, wherein the antistatic agent is crystallineascorbic acid.
 26. The method according to claim 1, wherein the mammalis a human having Alzheimer's disease.
 27. The method according to claim1, wherein the mammal is a human having Parkinson's disease.
 28. Themethod according to claim 1, wherein the mammal has acatecholamine-related toxicity.
 29. The method according to claim 1,wherein the mammal has malignant melanoma.
 30. The method according toclaim 1, wherein the mammal has atherosclerosis.
 31. The methodaccording to claim 1, wherein the mammal has macular degeneration. 32.The method according to claim 1, wherein the mammal has cataracts. 33.The method according to claim 1, wherein the mammal has glaucoma. 34.The method according to claim 1, wherein the mammal is a human havingadult respiratory distress syndrome (ARDS).
 35. The method according toclaim 1, wherein the mammal has emphysema.
 36. The method according toclaim 1, wherein the mammal has fibrocystic disease of the lung.
 37. Themethod according to claim 1, wherein the mammal has asbestosis.
 38. Themethod according to claim 1, wherein the mammal is a human havingAlzheimer's disease.
 39. The method according to claim 1, wherein theglutathione prevents malignant transformation of mammalian cells. 40.The method according to claim 1, wherein the mammal is poisoned with ametal ion.
 41. The method according to claim 40, wherein the metal ionis selected from the group consisting of cadmium, lead, mercury, copper,iron, selenium, tellurium, actinides and transuranics.
 42. The methodaccording to claim 1, wherein the mammal is subjected to ionizingradiation.
 43. The method according to claim 42, wherein the radiationis at a level above background levels.
 44. The method according to claim1, wherein the mammal is subjected to a toxic atmospheric gas.
 45. Themethod according to claim 44, wherein the toxic gas is selected from thegroup consisting of ozone, oxides of nitrogen, and oxides of sulfur. 46.The method according to claim 1, wherein the mammal has an inflammatorydisease of the bowel.
 47. The method according to claim 46, wherein theinflammatory disease is selected from the group consisting of regionalenteritis, and ulcerative colitis (Crohn's disease).
 48. The methodaccording to claim 1, wherein the mammal is administered a cancerchemotherapeutic agent.
 49. The method according to claim 48, whereinthe cancer chemotherapeutic agent is selected from the group consistingof cis-platin, doxorubicin, and daunorubicin.
 50. The method accordingto claim 1, wherein the mammal has suffered an acute injury.
 51. Themethod according to claim 50, wherein the injury is selected from thegroup consisting of spinal cord injury, brain injury, ophthalmic, andperipheral neuropathy.
 52. The method according to claim 1, wherein themammal suffers from halogenated hydrocarbon toxicity.
 53. A method ofinactivating virus in an extracorporeal human body fluid, comprisingadding reduced glutathione to the fluid in sufficient quantity to reduceviral proteins.
 54. The method according to claim 53, wherein the humanbody fluid comprises a blood product.
 55. A method of increasingglutathione levels in mammalian cells comprising orally administeringglutathione, in a formulation stabilized with an acidic antioxidantagent, to achieve an effective concentration in the duodenum of at leastabout 500 micromolar, with less than about 10 grams of food present pergram of glutathione in the duodenum.
 56. A method of administeringglutathione to a mammal, comprising maintaining substantially reducedL-glutathione and a reducing agent composition in a reduced condition ina pharmaceutical dosage form adapted to release at least a portion ofthe glutathione in the duodenum; and subsequently administering thepharmaceutical dosage form while the stomach is substantially empty,whereby absorption of the portion of glutathione released in theduodenum is greater than about 40%.
 57. A method of increasingglutathione levels in the tissues of a human, comprising the steps ofmaintaining a pharmaceutical formulation of substantially reducedL-glutathione mixed with a reducing agent composition in a reducedcondition and administering the pharmaceutical formulation in suchmanner to achieve a concentration of glutathione in the duodenumexceeding a concentration of glutathione in the cells lining theduodenum and as to promote conversion of less than about 10% ofadministered glutathione to ophthalmic acid in the stomach, duodenum andupper third of the ileum.
 58. A pharmaceutical formulation comprising adry gel matrix having therein glutathione or a derivative thereof, foradministration trans-mucosal membrane.
 59. The pharmaceuticalformulation according to claim 58, wherein said glutathione isderivatized as nitroso-glutathione.